Engineered phenylalanine ammonia lyase polypeptides

ABSTRACT

The present invention provides engineered phenylalanine ammonia lyase (PAL) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered phenylalanine ammonia lyase (PAL) polypeptides. Methods for producing PAL enzymes are also provided. In some embodiments, the engineered PAL polypeptides are optimized to provide enhanced catalytic activities that are useful under industrial process conditions for the production of pharmaceutical compounds.

The present application claims priority to co-pending U.S. patentapplication Ser. No. 16/441,458, filed on Jun. 14, 2019, which claimspriority to U.S. Pat. Appln. Ser. Nos. 62/696,978 and 62/814,362, filedon Jul. 12, 2018 and Mar. 6, 2019, respectively, all of which are herebyincorporated by reference in their entireties for all purposes.

FIELD OF THE INVENTION

The present invention provides engineered phenylalanine ammonia lyase(PAL) polypeptides and compositions thereof, as well as polynucleotidesencoding the engineered phenylalanine ammonia lyase (PAL) polypeptides.Methods for producing PAL enzymes are also provided. In someembodiments, the engineered PAL polypeptides are optimized to provideenhanced catalytic activities that are useful under industrial processconditions for the production of pharmaceutical compounds.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX2-179US1_ST25.txt”, a creation date of Jun. 12, 2019 anda size of 4.16 megabytes. The Sequence Listing filed via EFS-Web is partof the specification and incorporated in its entirety by referenceherein.

BACKGROUND OF THE INVENTION

Phenylalanine ammonia lyase (PAL) (See e.g., Cui et al., Crit. Rev.Biotechnol., 34: 258-268 [2014]; Hyun et al., Mycobiol., 39:257-265[2011]; MacDonald et al., Biochem. Cell Biol., 85:273-282 [2007]) alongwith histidine ammonia lyase (HAL) and tyrosine ammonia lyase (TAL) (Seee.g., Kyndt et al., FEBS Lett., 512:240-244 [2002]; Watt et al., Chem.Biol., 13: 1317-132 [2006]; and Xue et al., J. Ind. Microbiol.Biotechnol., 34:599-604 [2007]) are members of the aromatic amino acidlyase family (EC 4.3.1.23-1.25 and 4.3.1.3). More specifically, theenzymes having PAL activity (EC 4.3.1.23-1.25 and previously classifiedas EC 4.3.1.5) catalyze the nonoxidative deamination of L-phenylalanineinto (E)-cinnamic acid (Scheme 1).

The reaction is reversible and therefore PAL catalyzes the amination of(E)-cinnamic acid to give L-phenylalanine in the presence of highconcentrations of ammonia, or materials that liberate ammonia insolution (Scheme 1).

PAL is a non-mammalian enzyme that is widely distributed in plants andhas also been identified in fungi and a limited number of bacteria. Itis normally a tetramer with a typical mass of 300-340 kDa (See e.g., Cuiet al., Crit. Rev. Biotechnol., 34:258-268 [2014]). It does not requirean externally added cofactor, instead a cofactor is formed in the activesite of the enzyme via the self-cyclization and dehydration of threeresidues, namely, alanine, serine and glycine, to form3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) which acts as anelectrophile to catalyze the reaction (See e.g., MacDonald and D'Cunha,Biochem. Cell Biol., 85:273-82 [2007]).

Chiral amine compounds are frequently used in the pharmaceutical,agrochemical and chemical industries as intermediates or synthons forthe preparation of wide range of commercially desired compounds. It isestimated that 40% of current pharmaceuticals contain an aminefunctionality (See e.g., Ghislieri and Turner, Top. Catal., 57:284-300[2014].). Typically these industrial applications of chiral aminecompounds involve using only one particular stereomeric form of themolecule (e.g., only the (R) or the (S) enantiomer is physiologicallyactive).

PAL enzymes are highly enantioselective and have been used in theamination direction for the commercial synthesis of phenylalanine (Seee.g., Yamada et al., Appl. Environ. Microbiol., 19:421-427 [1981]; andEI-Batal et al., Acta Microbiol. Pol., 51:153-169 [2002]). EngineeredPAL variants have been reported with activity on cinnamic acids withsmall substituents on the aromatic ring (See e.g., Gloge et al. Chem.Eur. J., 6:3386-3390 [2000]; de Lange et al., Chem Cat Chem., 3:289-292[2011]; Lovelock et al., Bioorg. Med. Chem., 22:5555-5557 [2014];Parmeggiani et al., Angew. Chem. Int. Ed., 54:4608-4611 [2015]; Rowleset al., Tetrahed., 72:7343-7347 [2016]; and Weise et al., Catal. Sci.Technol., 6:4086-408 [2016])

Aqueous ammonia solutions adjusted with acid to the desired pH arecommonly used as the ammonia source when used in the amination reactiondirection for the production of a chiral amino acid. In addition,ammonia containing salts such as ammonium carbonate and ammoniumcarbamate (Weise et al., Catal. Sci. Technol., 6:4086-408 [2016])) havealso been used. Other ammonium salts can also be used such as ammoniumchloride, ammonium sulfate, ammonium acetate, ammonium phosphate,ammonium formate and others.

A major drawback of employing PALs commercially is they generally haveproperties that are undesirable for the preparation of industriallyimportant chiral amine compounds. These drawbacks include poor or noactivity on substrates containing bulky or electron rich substituent(s)on the aromatic ring compared to the native substrates cinnamic acid andphenylalanine, and often poor stability under industrially usefulprocess conditions (e.g., in the presence of organic solvents orelevated temperature). Thus, there is a need for engineered PALs thatcan be used in industrial processes for preparing chiral aminescompounds from a diverse range of substrates.

SUMMARY OF THE INVENTION

The present invention provides engineered phenylalanine ammonia lysate(PAL) enzymes, polypeptides having PAL activity, and polynucleotidesencoding these enzymes, as well as vectors and host cells comprisingthese polynucleotides and polypeptides. Methods for producing PALenzymes are also provided. The present invention further providescompositions comprising the PAL enzymes and methods of using theengineered PAL enzymes.

The present invention provides engineered phenylalanine ammonia lyase(PAL) polypeptides and compositions thereof, as well as polynucleotidesencoding the engineered phenylalanine ammonia lyase (PAL) polypeptides.In some embodiments, the engineered PAL polypeptides are optimized toprovide enhanced catalytic activities that are useful under industrialprocess conditions for the production of pharmaceutical compounds.

The present invention provides engineered phenylalanine ammonia lyasescomprising polypeptide sequences having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 2, 4, 8, 106, 252, 446, 482, 516, 618, 714, 830,894, and/or 988, or a functional fragment thereof. The present inventionalso provides engineered phenylalanine ammonia lyases comprisingpolypeptide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 2, 4, 8, 106, 252, 446, 482, 516, 618, 714, 830, 894, and/or 988,or a functional fragment thereof, wherein the engineered phenylalanineammonia lyases comprise at least one substitution or substitution set inthe polypeptide sequences, and wherein the amino acid positions of thepolypeptide sequences are numbered with reference to SEQ ID NO: 2, 4, 8,106, 252, 446, 482, 516, 618, 714, 830, 894, and/or 988, respectively.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 4, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 4, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set at one or more positions selected from80/99/104/175/220/359, 80/104, 80/104/105/172,80/104/105/172/175/222/359, 80/104/105/172/220/222, 80/104/105/220,80/104/105/220/222/416, 80/104/105/222, 80/104/172/175,80/104/172/175/220/310/359, 80/104/172/222, 80/104/359/416, 84, 90,99/104/105/172/175/220/222, 100, 101, 104, 104/105/175, 104/172/310/359,104/175/213/222/359, 104/175/220/222, 104/220/222/359, 104/359, 107,108, 110/419, 175/315, 219, 219/540, 220, 347, 360, 363, 405, 416, 418,423, 450, 451, and 452, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 4. Insome additional embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 4, or a functional fragment thereof, and whereinthe engineered phenylalanine ammonia lyase comprises at least onesubstitution or substitution set selected from80A/99D/104A/175A/220G/359Y, 80A/104A, 80A/104A/105I/172A,80A/104A/105I/172A/220G/222V, 80A/104A/105I/172T/175A/222V/359Y,80A/104A/105I/220G, 80A/104A/105I/220G/222V/416V, 80A/104A/105I/222V,80A/104A/172A/175A, 80A/104A/172A/175A/220G/310A/359Y,80A/104A/172T/222V, 80A/104A/359Y/416V, 84P, 84V, 90T,99D/104A/105I/172I/175A/220G/222V, 100R, 100S, 101K, 104A,104A/105I/175A, 104A/172A/310A/359Y, 104A/175A/213Q/222V/359Y,104A/175A/220G/222V, 104A/220G/222V/359Y, 104A/359Y, 104I, 104P, 104S,107T, 108E, 110P/419D, 175G/315R, 219G, 219M/540G, 219P, 220P, 347V,360V, 363R, 405R, 416E, 416L, 418G, 423E, 450E, 451P, and 452A, whereinthe amino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 4. In some additional embodiments, theengineered phenylalanine ammonia lyase comprises a polypeptide sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4, or afunctional fragment thereof, and wherein the engineered phenylalanineammonia lyase comprises at least one substitution or substitution setselected from V80A/E99D/L104A/S175A/A220G/H359Y, V80A/L104A,V80A/L104A/V105I/V172A, V80A/L104A/V105I/V172A/A220G/M222V,V80A/L104A/V105I/V172T/S175A/M222V/H359Y, V80A/L104A/V105I/A220G,V80A/L104A/V105I/A220G/M222V/M416V, V80A/L104A/V105I/M222V,V80A/L104A/V172A/S175A, V80A/L104A/V172A/S175A/A220G/I310A/H359Y,V80A/L104A/V172T/M222V, V80A/L104A/H359Y/M416V, F84P, F84V, V90T,E99D/L104A/V105I/V172T/S175A/A220G/M222V, L100R, L100S, Q101K, L104A,L104A/V105I/S175A, L104A/V172A/I310A/H359Y,L104A/S175A/L213Q/M222V/H359Y, L104A/S175A/A220G/M222V,L104A/A220G/M222V/H359Y, L104A/H359Y, L104I, L104P, L104S, H107T, L108E,T110P/K419D, S175G/S315R, L219G, L219M/E540G, L219P, A220P, N347V,G360V, F363R, S405R, M416E, M416L, L418G, 1423E, F450E, N451P, andQ452A, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 4.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 8, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 8, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set at one or more positions selected from 20/306/564,74/80/105/107/394/420, 74/83/102/105/107/111/222/394/416,74/97/105/106/107, 74/102/105/106/107/175/394,74/102/105/106/107/175/394/421, 80/84/99/104/105/107/219,80/102/105/107/304, 83/102/105/106/107/416/420/421,83/102/105/394/416/420, 84, 84/99, 84/99/104/105/219, 84/107,97/102/105/106/107/111/394/420/421, 97/102/105/107/111/175/304/421/424,97/102/111/175/222/420/421, 97/105/107/111/222/421/424,97/105/107/111/394/416/421, 99/105/107, 102,102/105/107/222/304/307/394/421/424, 102/105/107/222/304/394/421/424,102/105/107/304/424, 102/105/107/394/416/424, 102/107/111/222/394,102/107/420/424, 103, 104, 105, 105/106/107/420/421, 105/107,105/107/111, 105/107/111/304, 105/107/111/394/420/424,105/107/222/304/416, 105/111/219, 105/175/219, 105/219, 106, 107,107/111/209/222/304, 107/222/304, 107/291, 107/421, 175, 216, 219, 220,222/421/424, 304/394/416/420, 306, 359, 394, 395, 413, 416, 418, and420, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 8. In some additional embodiments,the engineered phenylalanine ammonia lyase comprises a polypeptidesequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 8,or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set selected from 20D/306L/564Q,74D/80A/105I/107G/394N/420S, 74D/83S/102E/105I/107S/111A/222A/394N/416V,74D/97T/105I/106R/107G, 74D/102E/105I/106R/107G/175A/394N/421T,74D/102E/105I/106R/107L/175A/394N, 80A/84V/99D/104I/105I/107T/219G,80A/102E/105I/107L/304W, 83S/102E/105I/106R/107G/416V/420S/421T,83S/102E/105I/394N/416V/420S, 84G, 84L, 84P, 84R, 84S, 84V, 84V/99D,84V/99D/104I/105I/219G, 84V/107T,97T/102E/105I/106R/107G/111A/394N/420S/421T,97T/102E/105I/107G/111A/175A/304W/421T/424V,97T/102E/111A/175A/222G/420S/421T, 97T/105I/107G/111A/222G/421T/424V,97T/105I/107S/111A/394N/416V/421T, 99D/105I/107T,102E/105I/107G/394N/416V/424V, 102E/105I/107I/304W/424V,102E/105I/107S/222G/304W/307H/394N/421T/424V,102E/105I/107S/222G/304W/394N/421T/424V, 102E/107A/111A/222G/394N,102E/107G/420S/424V, 102N, 103S, 104G, 105I, 105I/106R/107G/420S/421T,105I/107A/222G/304W/416V, 105I/107E/111A, 105I/107G/111A/394N/420S/424V,105I/107I/111A/304W, 105I/107T, 105I/111A/219G, 105I/175A/219G,105I/219G, 106M, 107G, 107G/291N, 107G/421T, 107L, 107L/222G/304W, 107P,107Q, 107T, 107T/111A/209I/222G/304W, 175N, 216G, 219C, 220S,222A/421T/424V, 304W/394N/416V/420S, 306L, 359R, 394V, 395M, 413E, 413T,416A, 416C, 416G, 416H, 416L, 416V, 418I, 420A, and 420S, wherein theamino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 8. In some additional embodiments, theengineered phenylalanine ammonia lyase comprises a polypeptide sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 8, or afunctional fragment thereof, and wherein the engineered phenylalanineammonia lyase comprises at least one substitution or substitution setselected from G20D/D306L/P564Q, G74D/V80A/V105I/H107G/A394N/G420S,G74D/G83S/T102E/V105I/H107S/G111A/V222A/A394N/M416V,G74D/A97T/V105I/W106R/H107G,G74D/T102E/V105I/W106R/H107G/S175A/A394N/L421T,G74D/T102E/V105I/W106R/H107L/S175A/A394N,V80A/F84V/E99D/A104I/V105I/H107T/L219G, V80A/T102E/V105I/H107L/Y304W,G83S/T102E/V105I/W106R/H107G/M416V/G420S/L421T,G83S/T102E/V105I/A394N/M416V/G420S, F84G, F84L, F84P, F84R, F84S, F84V,F84V/E99D, F84V/E99D/A104I/V105I/L219G, F84V/H107T,A97T/T102E/V105I/W106R/H107G/G111A/A394N/G420S/L421T,A97T/T102E/V105I/H107G/G111A/S175A/Y304W/L421T/C424V,A97T/T102E/G111A/S175A/V222G/G420S/L421T,A97T/V105I/H107G/G111A/V222G/L421T/C424V,A97T/V105I/H107S/G111A/A394N/M416V/L421T, E99D/V105I/H107T,T102E/V105I/H107G/A394N/M416V/C424V, T102E/V105I/H107I/Y304W/C424V,T102E/V105I/H107S/V222G/Y304W/G307H/A394N/L421T/C424V,T102E/V105I/H107S/V222G/Y304W/A394N/L421T/C424V,T102E/H107A/G111A/V222G/A394N, T102E/H107G/G420S/C424V, T102N, N103S,A104G, V105I, V105I/W106R/H107G/G420S/L421T,V105I/H107A/V222G/Y304W/M416V, V105I/H107E/G111A,V105I/H107G/G111A/A394N/G420S/C424V, V105I/H107I/G111A/Y304W,V105I/H107T, V105I/G111A/L219G, V105I/S175A/L219G, V105I/L219G, W106M,H107G, H107G/5291N, H107G/L421T, H107L, H107L/V222G/Y304W, H107P, H107Q,H107T, H107T/G111A/S209I/V222G/Y304W, S175N, K216G, L219C, G220S,V222A/L421T/C424V, Y304W/A394N/M416V/G420S, D306L, Y359R, A394V, S395M,K413E, K413T, M416A, M416C, M416G, M416H, M416L, M416V, L418I, G420A,and G420S, wherein the amino acid positions of the polypeptide sequenceare numbered with reference to SEQ ID NO: 8.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 106, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 106, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set at one or more positions selected from 3, 3/550, 4, 5,6, 7, 10, 14, 22, 22/76, 24, 25, 40, 75, 76, 76/561, 84/107/175/219,84/107/219, 102/107/219/410, 107, 107/216/410, 107/219, 107/220, 212,219/220, 219/220/410, 220/359, 220/410, 286, 301, 303, 410, 502, 544,566, and 567, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 106. In someadditional embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 106, or a functional fragment thereof, andwherein the engineered phenylalanine ammonia lyase comprises at leastone substitution or substitution set selected from 3E, 3E/550T, 3H, 3K,3N, 3P, 3R, 4P, 4S, 5D, 5L, 5P, 6D, 6S, 7D, 7G, 7T, 10A, 10P, 10S, 14A,22A, 22V/76S, 24V, 25R, 40D, 75L, 76A, 76E, 76H, 76L, 76L/561L, 76M,76R, 76T, 84P/107A/175A/219C, 84P/107A/219C, 84P/107G/219C,102N/107G/219C/410K, 107A, 107A/219C, 107G, 107G/216G/410K, 107G/219C,107G/220S, 212N, 212P, 219C/220S, 219C/220S/410K, 220S/359R, 220S/410K,286R, 301S, 303I, 303K, 303R, 303T, 303V, 410K, 502Q, 502T, 544W, 566G,and 567D, wherein the amino acid positions of the polypeptide sequenceare numbered with reference to SEQ ID NO: 106. In some additionalembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 106, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set selected from T3E, T3E/A550T, T3H, T3K, T3N, T3P, T3R,L4P, L4S, S5D, S5L, S5P, Q6D, Q6S, A7D, A7G, A7T, K10A, K10P, K10S,Q14A, S22A, S22V/P76S, A24V, N25R, R40D, E75L, P76A, P76E, P76H, P76L,P76L/D561L, P76M, P76R, P76T, F84P/S107A/S175A/L219C, F84P/S107A/L219C,F84P/S107G/L219C, E102N/S107G/L219C/R410K, S107A, S107A/L219C, S107G,S107G/K216G/R410K, S107G/L219C, S107G/G220S, T212N, T212P, L219C/G220S,L219C/G220S/R410K, G220S/Y359R, G220S/R410K, S286R, K301S, D303I, D303K,D303R, D303T, D303V, R410K, A502Q, A502T, R544W, L566G, and H567D,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 106.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 252, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 252, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set at one or more positions selected from3/4/5/7/76/84/107/307, 3/5/107/307/566, 3/7/76/107/307/566, 3/7/84/307,24/76/107/307, 24/84/107/307, 76, 76/84/107, 76/84/107/307,76/84/107/307/502, 76/84/107/502, 76/107, 76/107/307, 76/307,76/307/502, 84/107/307, 84/107/307/502, 84/301/307/566, 84/307,107/301/502, 107/307, 107/307/566, 107/502, 107/502/566, 307, 307/502,and 307/566, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 252. In someadditional embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 252, or a functional fragment thereof, andwherein the engineered phenylalanine ammonia lyase comprises at leastone substitution or substitution set selected from3E/4S/5P/7G/76T/84P/107A/307G, 3E/7G/84P/307G, 3K/7D/76L/107A/307G/566G,3P/5P/107A/307G/566G, 24V/76A/107A/307G, 24V/84P/107G/307G,76A/84P/107A/502Q, 76H/84P/107A, 76H/307G/502Q, 76L/107A/307G,76L/107G/307G, 76L/307G, 76M/84P/107A, 76M/84P/107A/307G, 76M/84P/107G,76M/84P/107G/307G, 76M/107A, 76M/107G, 76T, 76T/84P/107A/307G/502Q,76T/84P/107G, 76T/107G, 84P/107A/307G, 84P/107A/307G/502Q,84P/301S/307G/566G, 84P/307G, 107A/301S/502Q, 107A/307G, 107A/307G/566G,107A/502Q, 107A/502Q/566G, 307G, 307G/502Q, and 307G/566G, wherein theamino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 252. In some additional embodiments, theengineered phenylalanine ammonia lyase comprises a polypeptide sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 252, or afunctional fragment thereof, and wherein the engineered phenylalanineammonia lyase comprises at least one substitution or substitution setselected from T3E/L4S/S5P/A7G/P76T/F84P/S107A/H307G, T3E/A7G/F84P/H307G,T3K/A7D/P76L/S107A/H307G/L566G, T3P/S5P/S107A/H307G/L566G,A24V/P76A/S107A/H307G, A24V/F84P/S107G/H307G, P76A/F84P/S107A/A502Q,P76H/F84P/S107A, P76H/H307G/A502Q, P76L/S107A/H307G, P76L/S107G/H307G,P76L/H307G, P76M/F84P/S107A, P76M/F84P/S107A/H307G, P76M/F84P/S107G,P76M/F84P/S107G/H307G, P76M/S107A, P76M/S107G, P76T,P76T/F84P/S107A/H307G/A502Q, P76T/F84P/S107G, P76T/S107G,F84P/S107A/H307G, F84P/S107A/H307G/A502Q, F84P/K301S/H307G/L566G,F84P/H307G, S107A/K301S/A502Q, S107A/H307G, S107A/H307G/L566G,S107A/A502Q, S107A/A502Q/L566G, H307G, H307G/A502Q, and H307G/L566G,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 252.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 446, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 446, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set at one or more positions selected from 3/4/6/7/76,3/4/6/7/303, 3/4/7, 3/4/7/76, 3/6/502, 3/7/76, 7/303, 40/303, 76,76/502, 82, 100, 102, 171, 174, 216, 218, 219, 222, 222/509, 303,303/502, 304, and 345, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 446. Insome additional embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 446, or a functional fragment thereof, andwherein the engineered phenylalanine ammonia lyase comprises at leastone substitution or substitution set selected from 3E/4S/7V/76H,3E/6S/502Q, 3H/4S/7G, 3H/7D/76H, 3H/7D/76R, 3K/4S/6S/7V/303I,3T/4S/6D/7D/76R, 7G/303I, 40D/303I, 76A, 76H, 76L, 76R, 76R/502Q, 82T,100H, 102M, 171P, 171V, 174G, 216G, 218A, 219M, 219T, 222T, 222T/509K,222V, 303I, 303I/502Q, 304F, 304H, 304V, and 345S, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 446. In some additional embodiments, the engineeredphenylalanine ammonia lyase comprises a polypeptide sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NO: 446, or a functionalfragment thereof, and wherein the engineered phenylalanine ammonia lyasecomprises at least one substitution or substitution set selected fromP3E/L4S/A7V/P76H, P3E/Q6S/A502Q, P3H/L4S/A7G, P3H/A7D/P76H,P3H/A7D/P76R, P3K/L4S/Q6S/A7V/D303I, P3T/L4S/Q6D/A7D/P76R, A7G/D303I,R40D/D303I, P76A, P76H, P76L, P76R, P76R/A502Q, S82T, L100H, E102M,L171P, L171V, L174G, K216G, G218A, C219M, C219T, G222T, G222T/E509K,G222V, D303I, D303I/A502Q, W304F, W304H, W304V, and T345S, wherein theamino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 446.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 482, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 482, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set at one or more positions selected from 3/4/7, 3/7, 4/7,4/7/216/218, 7, 7/40, 7/76/82/174/222/303, 7/76/174/222, 7/76/219/303,7/82, 7/216/218, 40/82, 47, 66, 76/82/216/218, 76/216, 76/216/219, 82,112, 171, 174/222, 209, 216, 219, 219/345, 222, 268, 271, 331, 366, 428,437, 443, 460, 474, 503, 524, 538, and 543, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 482. In some additional embodiments, the engineered phenylalanineammonia lyase comprises a polypeptide sequence having at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 482, or a functional fragment thereof,and wherein the engineered phenylalanine ammonia lyase comprises atleast one substitution or substitution set selected from 3E/4S/7D,3E/7D, 4S/7D, 4S/7D/216G/218A, 7D, 7D/40D, 7D/76L/82T/174G/222T/303I,7D/76M/174G/222V, 7D/76M/219T/303I, 7D/82T, 7D/216G/218A, 40D/82T, 47P,47Q, 66W, 76L/82T/216G/218A, 76M/216G, 76M/216G/219T, 82T, 112L, 112S,112T, 171P, 174G/222V, 209A, 216G, 219T, 219T/3455, 222V, 268T, 271A,331T, 366S, 428L, 428M, 437H, 443Q, 460F, 474Y, 503T, 524A, 524D, 524R,538I, 538V, and 543Q, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 482. Insome additional embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 482, or a functional fragment thereof, andwherein the engineered phenylalanine ammonia lyase comprises at leastone substitution or substitution set selected from P3E/L4S/A7D, P3E/A7D,L4S/A7D, L4S/A7D/K216G/G218A, A7D, A7D/R40D,A7D/P76L/S82T/L174G/G222T/D303I, A7D/P76M/L174G/G222V,A7D/P76M/C219T/D303I, A7D/S82T, A7D/K216G/G218A, R40D/S82T, L47P, L47Q,Y66W, P76L/S82T/K216G/G218A, P76M/K216G, P76M/K216G/C219T, S82T, A112L,A1125, A112T, L171P, L174G/G222V, S209A, K216G, C219T, C219T/T3455,G222V, I268T, S271A, S331T, Q3665, I428L, I428M, N437H, F443Q, T460F,N474Y, C503T, S524A, S524D, S524R, L538I, L538V, and A543Q wherein theamino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 482.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 516, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 516, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set at one or more positions selected from 4, 4/304, 6, 7,9, 16, 20, 25, 40, 40/437, 44/47, 44/47/94/509, 44/94/270/554,44/94/554, 47, 47/76, 47/76/345, 47/94/509, 47/195/554, 47/428, 51/106,76, 76/271, 76/345, 82, 84, 94/149, 94/195, 94/554, 98, 98/460, 109,112/524, 271/345, 271/428, 302, 303, 304, 306, 349, 358, 410, 413, 416,and 524, wherein the amino acid positions of the polypeptide sequenceare numbered with reference to SEQ ID NO: 516. In some additionalembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO:516, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set selected from 4G, 4I/304C, 6R, 7S, 9C, 16S, 16Y, 20T,25A, 25G, 40D, 40D/437H, 44H/47K, 44H/47K/94P/509L, 44H/94P/270Q/554R,44H/94P/554R, 47K, 47K/94P/509L, 47K/195E/554R, 47P, 47P/76H,47P/76H/345S, 47P/428L, 51A/106G, 76H, 76H/271A, 76H/345S, 82T, 84P,94P/149T, 94P/195E, 94P/554R, 98A, 98E, 98N/460A, 109G, 112L/524A,271A/345S, 271A/428L, 302R, 303I, 304H, 304L, 304S, 306K, 349I, 358L,410M, 413S, 413T, 416T, and 524A, wherein the amino acid positions ofthe polypeptide sequence are numbered with reference to SEQ ID NO: 516.In some additional embodiments, the engineered phenylalanine ammonialyase comprises a polypeptide sequence having at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 516, or a functional fragment thereof,and wherein the engineered phenylalanine ammonia lyase comprises atleast one substitution or substitution set selected from L4G, L4I/W304C,Q6R, D7S, S9C, F165, F16Y, G20T, N25A, N25G, R40D, R40D/N437H,N44H/L47K, N44H/L47K/R94P/E509L, N44H/R94P/N270Q/V554R, N44H/R94P/V554R,L47K, L47K/R94P/E509L, L47K/K195E/V554R, L47P, L47P/M76H,L47P/M76H/T345S, L47P/I428L, T51A/W106G, M76H, M76H/S271A, M76H/T3455,S82T, F84P, R94P/I149T, R94P/K195E, R94P/V554R, S98A, S98E, S98N/T460A,K109G, A112L/S524A, S271A/T345S, S271A/I428L, H302R, D303I, W304H,W304L, W3045, D306K, L349I, Y358L, R410M, K4135, K413T, M416T, andS524A, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 516.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 618, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 618, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set at one or more positions selected from 4/47/76/82/94,7/44/94/98, 7/47/76/82, 7/76/554, 7/94/98, 16/40/76, 16/44/76/98,16/44/94/98/524, 25/54/68/72, 25/54/68/72/158/339,25/54/68/72/209/212/339/517, 25/54/68/158/339/517, 25/54/72/339/517,25/54/158/209/212/339/551, 30/68/72/207/209/212/339/495/517,40/44/76/304/509, 40/44/98/304, 40/76/304/437, 40/76/554,44/76/94/112/304, 44/76/112, 44/94/271/304/437/554, 47/76/82/94/271,47/76/82/271/304, 47/76/94/271, 47/76/94/271/306/375/524/554,47/76/304/306/554, 47/76/304/524/554, 47/94, 47/94/271,47/94/271/304/554, 49/114/240/521, 54/68/158/209/212/495/517,68/72/158/209/212/339/495/551, 68/72/158/517, 68/158/209/495/517/551,76, 76/271/304/554, 76/304/437, 82, 82/554, 94/98, 94/98/306, 94/98/509,94/98/524, 94/554, 98/270/304/554, 119, 294, 357, 400, 516, 527, and565, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 618. In some additionalembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 618, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set selected from 4I/47K/76H/82T/94P, 7S/44H/94P/98N,7S/47P/76H/82T, 7S/76H/554R, 7S/94P/98E, 16Y/40D/76H, 16Y/44H/76H/98E,16Y/44H/94P/98E/524A, 25T/54E/68A/72A/158H/339V,25T/54E/68A/72A/209E/212Q/339V/517E, 25T/54E/72A/339V/517E,25T/54E/158H/209E/212Q/339V/551S, 25T/54K/68A/72A,25T/54K/68A/158H/339V/517E, 30G/68A/72A/207G/209E/212Q/339V/495A/517E,40D/44H/76H/304H/509L, 40D/44H/98A/304S, 40D/76H/304S/437H,40D/76H/554R, 44H/76H/94P/112L/3045, 44H/76H/112L,44H/94P/271A/304S/437H/554R, 47K/76H/82T/94P/271A,47K/76H/82T/271A/304S, 471K/76H/94P/271A, 471K/76H/3045/306K/554R,471K/94P, 471K/94P/271A, 471K/94P/271A/3045/554R,47P/76H/94P/271A/306K/375M/524A/554R, 47P/76H/304S/524A/554R,49R/114K/240K/521K, 54K/68A/158H/209E/212Q/495A/517E,68A/72A/158H/209E/212Q/339V/495A/5515, 68A/72A/158H/517E,68A/158H/209E/495A/517E/551S, 76H, 76H/271A/304S/554R, 76H/304S/437H,82T, 82T/554R, 94P/98E/306K, 94P/98E/509L, 94P/98E/524A, 94P/98N,94P/554R, 98E/270Q/304S/554R, 119E, 119V, 294A, 294C, 357I, 400A, 516M,527V, and 565E, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 618. In someadditional embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 618, or a functional fragment thereof, andwherein the engineered phenylalanine ammonia lyase comprises at leastone substitution or substitution set selected fromL4I/L47K/M76H/S82T/R94P, D7S/N44H/R94P/S98N, D7S/L47P/M76H/S82T,D7S/M76H/V554R, D7S/R94P/S98E, F16Y/R40D/M76H, F16Y/N44H/M76H/S98E,F16Y/N44H/R94P/S98E/S524A, N25T/T54E/N68A/E72A/Y158H/I339V,N25T/T54E/N68A/E72A/S209E/T212Q/I339V/H517E, N25T/T54E/E72A/I339V/H517E,N25T/T54E/Y158H/S209E/T212Q/I339V/A551S, N25T/T54K/N68A/E72A,N25T/T54K/N68A/Y158H/I339V/H517E,N30G/N68A/E72A/N207G/S209E/T212Q/I339V/T495A/H517E,R40D/N44H/M76H/W304H/E509L, R40D/N44H/S98A/W304S, R40D/M76H/W304S/N437H,R40D/M76H/V554R, N44H/M76H/R94P/A112L/W304S, N44H/M76H/A112L,N44H/R94P/S271A/W304S/N437H/V554R, L47K/M76H/S82T/R94P/S271A,L47K/M76H/S82T/S271A/W304S, L47K/M76H/R94P/S271A,L47K/M76H/W304S/D306K/V554R, L47K/R94P, L47K/R94P/S271A,L47K/R94P/S271A/W304S/V554R,L47P/M76H/R94P/S271A/D306K/L375M/S524A/V554R,L47P/M76H/W304S/S524A/V554R, S49R/N114K/Q240K/Q521K,T54K/N68A/Y158H/S209E/T212Q/T495A/H517E,N68A/E72A/Y158H/S209E/T212Q/I339V/T495A/A551S, N68A/E72A/Y158H/H517E,N68A/Y158H/S209E/T495A/H517E/A551S, M76H, M76H/S271A/W304S/V554R,M76H/W304S/N437H, S82T, S82T/V554R, R94P/S98E/D306K, R94P/S98E/E509L,R94P/S98E/S524A, R94P/S98N, R94P/V554R, S98E/N270Q/W304S/V554R, A119E,A119V, V294A, V294C, S357I, N400A, R516M, R527V, and C565E, wherein theamino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 618.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 714, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 714, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set at one or more positions selected from 25,25/40/158/209/304/410/517, 25/54/271/517, 25/158/209/410, 25/306/339,25/410, 40/47/68/94/98/410/517, 40/68/460/517, 47/98/339/410,54/68/72/98/209/517, 68, 68/339/517, 72/94/158/339/410/460/517,72/158/209/410/517, 83, 94/158/209/339/410, 100, 129, 158,158/207/339/410, 158/209/410/517, 207/410, 207/410/460/517, 220, 317,339, 394, 410, 410/517, 416, 460, 460/517, and 517, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 714. In some additional embodiments, the engineeredphenylalanine ammonia lyase comprises a polypeptide sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NO: 714, or a functionalfragment thereof, and wherein the engineered phenylalanine ammonia lyasecomprises at least one substitution or substitution set selected from25T, 25T/40D/158H/209E/304H/410M/517E, 25T/54E/271A/517E,25T/158H/209E/410M, 25T/306E/339V, 25T/410M,40D/47K/68A/94P/98A/410M/517E, 40D/68A/460A/517E, 47K/98E/339V/410M,54E/68A/72A/98E/209E/517E, 68A, 68A/339V/517E,72A/94P/158H/339V/410M/460A/517E, 72A/158H/209E/410M/517E, 83L, 83P,94P/158H/209E/339V/410M, 100G, 1291, 158H, 158H/207G/339V/410M,158H/209E/410M/517E, 207G/410M, 207G/410M/460A/517E, 220A, 317E, 339V,394S, 410M, 410M/517E, 416I, 460A, 460A/517E, and 517E, wherein theamino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 714. In some additional embodiments, theengineered phenylalanine ammonia lyase comprises a polypeptide sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 714, or afunctional fragment thereof, and wherein the engineered phenylalanineammonia lyase comprises at least one substitution or substitution setselected from N25T, N25T/R40D/Y158H/S209E/S304H/R410M/H517E,N25T/T54E/S271A/H517E, N25T/Y158H/S209E/R410M, N25T/D306E/I339V,N25T/R410M, R40D/P47K/N68A/R94P/S98A/R410M/H517E, R40D/N68A/T460A/H517E,P47K/S98E/I339V/R410M, T54E/N68A/E72A/S98E/S209E/H517E, N68A,N68A/I339V/H517E, E72A/R94P/Y158H/I339V/R410M/T460A/H517E,E72A/Y158H/S209E/R410M/H517E, G83L, G83P, R94P/Y158H/S209E/I339V/R410M,L100G, A1291, Y158H, Y158H/N207G/I339V/R410M, Y158H/S209E/R410M/H517E,N207G/R410M, N207G/R410M/T460A/H517E, S220A, R317E, I339V, N394S, R410M,R410M/H517E, M416I, T460A, T460A/H517E, and H517E, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 714.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 830, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 830, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set at one or more positions selected from N25T,N25T/R40D/Y158H/S209E/S304H/R410M/H517E, N25T/T54E/S271A/H517E,N25T/Y158H/S209E/R410M, N25T/D306E/I339V, N25T/R410M,R40D/P47K/N68A/R94P/S98A/R410M/H517E, R40D/N68A/T460A/H517E,P47K/S98E/I339V/R410M, T54E/N68A/E72A/S98E/S209E/H517E, N68A,N68A/I339V/H517E, E72A/R94P/Y158H/I339V/R410M/T460A/H517E,E72A/Y158H/S209E/R410M/H517E, G83L, G83P, R94P/Y158H/S209E/I339V/R410M,L100G, A1291, Y158H, Y158H/N207G/I339V/R410M, Y158H/S209E/R410M/H517E,N207G/R410M, N207G/R410M/T460A/H517E, S220A, R317E, I339V, N394S, R410M,R410M/H517E, M416I, T460A, T460A/H517E, and H517E, wherein the aminoacid positions of the polypeptide sequence are numbered with referenceto SEQ ID NO: 830. In some additional embodiments, the engineeredphenylalanine ammonia lyase comprises a polypeptide sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NO: 830, or a functionalfragment thereof, and wherein the engineered phenylalanine ammonia lyasecomprises at least one substitution or substitution set selected from25T/83L/158H/220A/517E, 25T/83P/220A/416I, 25T/158H/209E/220A/517E,25T/158H/220A, 25T/158H/220A/517E, 25T/220A/339V, 25T/220A/517E,25T/410M/416I/517E, 40G, 45H, 54K/59R, 54K/285L, 83P,83P/209E/220A/410M/517E, 83P/339V/410M, 119Q, 158H/220A/271A/517E, 209P,209T, 220A, 220A/410M/416I/517E, 220A/517E, 244S, 246V, 271A,271A/410M/416I/517E, 293M, 304A, 339V, 368F, 400A, 400Q, 410A, 410E,410M/416I/517E, 424A, 459F, 479S, 520A, 525P, 537A, 537P, 562V, and565K, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 830. In some additionalembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 830, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set selected from N25T/G83L/Y158H/S220A/H517E,N25T/G83P/S220A/M416I, N25T/Y158H/S209E/S220A/H517E, N25T/Y158H/S220A,N25T/Y158H/S220A/H517E, N25T/S220A/I339V, N25T/S220A/H517E,N25T/R410M/M416I/H517E, R40G, G45H, T54K/G59R, T54K/I285L, G83P,G83P/S209E/S220A/R410M/H517E, G83P/I339V/R410M, A119Q,Y158H/S220A/S271A/H517E, S209P, S209T, S220A, S220A/R410M/M416I/H517E,S220A/H517E, A244S, A246V, S271A, S271A/R410M/M416I/H517E, L293M, S304A,I339V, V368F, N400A, N400Q, R410A, R410E, R410M/M416I/H517E, V424A,Y459F, A479S, G520A, 5525P, G537A, G537P, I562V, and C565K, wherein theamino acid positions of the polypeptide sequence are numbered withreference to SEQ ID NO: 830.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 894, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 894, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set at one or more positions selected from25/40/45/209/424, 25/40/424, 25/45/54/73/246/424, 25/54/73/209/424/520,40/47/54/214/503, 40/54/209/214/244/339/520, 40/54/214/244/339/503,40/209/246/424, 54, 54/209/214/244, 54/209/214/244/339/503, 54/424,54/424/520, 209/503, 227, 246, 246/424, 274/311, 410, 411, 413, and 424,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 894. In some additionalembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 894, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set selected from 25T/40C/424A, 25T/40G/45H/209P/424A,25T/45H/54L/73K/246V/424A, 25T/54P/73K/209T/424A/520A,40G/209P/246V/424A, 40Q/47R/54P/214N/503T,40Q/54P/209P/214N/2445/339V/520A, 40T/54P/214N/244S/339V/503T,54K/209P/214N/244S/339V/503T, 54P, 54P/209P/214N/244S, 54P/424A,54P/424A/520A, 209P/503T, 227F, 246V, 246V/424A, 274P/311S, 410Q, 411A,413S, 424A, 424C, 424G, and 424S, wherein the amino acid positions ofthe polypeptide sequence are numbered with reference to SEQ ID NO: 894.In some additional embodiments, the engineered phenylalanine ammonialyase comprises a polypeptide sequence having at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity to SEQ ID NO: 894, or a functional fragment thereof,and wherein the engineered phenylalanine ammonia lyase comprises atleast one substitution or substitution set selected fromN25T/R40C/V424A, N25T/R40G/G45H/E209P/V424A,N25T/G45H/T54L/S73K/A246V/V424A, N25T/T54P/S73K/E209T/V424A/G520A,R40G/E209P/A246V/V424A, R40Q/P47R/T54P/L214N/C503T,R40Q/T54P/E209P/L214N/A244S/I339V/G520A,R40T/T54P/L214N/A244S/I339V/C503T, T54K/E209P/L214N/A244S/I339V/C503T,T54P, T54P/E209P/L214N/A244S, T54P/V424A, T54P/V424A/G520A, E209P/C503T,V227F, A246V, A246V/V424A, H274P/Q311S, M410Q, E411A, K413S, V424A,V424C, V424G, and V424S, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 894.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 988, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 988, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set at one or more positions selected from 40,40/54/214/421/424, 40/90/421/424, 40/214, 40/214/424, 40/421/424,40/424, 54/214, 66, 90/214/424, 106/227, 106/227/244, 106/227/244/554,106/227/554, 214, 214/421, 339, 421/424, 424, 454, 463, 464, 474, and543, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 988. In some additionalembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 988, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set selected from 40C, 40C/54L/214Q/4215/424C,40C/90Q/421S/424C, 40C/214N/424C, 40C/214Q, 40C/421S/424G, 40C/424C,54L/214Q, 66F, 90Q/214Q/424G, 106R/227F, 106S/227F/244S,106S/227F/244S/554C, 106S/227F/554C, 214Q, 214Q/421S, 339M, 421S/424C,424C, 454L, 454V, 463A, 463G, 463L, 463N, 463S, 463V, 463W, 464C, 464Q,474E, and 543Q, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 988. In someadditional embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 988, or a functional fragment thereof, andwherein the engineered phenylalanine ammonia lyase comprises at leastone substitution or substitution set selected from R40C,R40C/P54L/L214Q/T421S/A424C, R40C/V90Q/T421S/A424C, R40C/L214N/A424C,R40C/L214Q, R40C/T421S/A424G, R40C/A424C, P54L/L214Q, Y66F,V90Q/L214Q/A424G, W106R/V227F, W106S/V227F/A244S,W106S/V227F/A244S/R554C, W106S/V227F/R554C, L214Q, L214Q/T421S, I339M,T421S/A424C, A424C, I454L, I454V, T463A, T463G, T463L, T463N, T463S,T463V, T463W, L464C, L464Q, N474E, and A543Q, wherein the amino acidpositions of the polypeptide sequence are numbered with reference to SEQID NO: 988.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 988, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 988, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set at one or more positions selected from 36, 40/66/227,40/66/227/244/410/424, 40/66/410/474, 40/410/411/424, 47, 66,66/214/374/410/474, 66/214/424, 66/214/437/474, 66/227,66/227/244/424/543, 66/227/424, 66/339, 66/339/410/543, 66/339/474,66/370, 66/410/424/454/527, 66/424, 66/463/464, 66/543, 102, 104, 105,154, 214/244/543, 214/374/424, 227, 227/244/411/424, 227/339/413/437,244/411, 339, 394, 410, 410/411/424, 413, 421, 424, 517, 524, and 554,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 988. In some additionalembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 988, or a functional fragment thereof, and wherein the engineeredphenylalanine ammonia lyase comprises at least one substitution orsubstitution set selected from 36C, 36Q, 40C/66F/227F,40C/66F/227F/244S/410K/424C, 40C/66F/410K/474E, 40C/410K/411A/424C, 47A,47E, 47R, 47T, 66F, 66F/214Q/374D/410K/474E, 66F/214Q/424C,66F/214Q/437G/474E, 66F/227F, 66F/227F/244S/424C/543Q, 66F/227F/424G,66F/339L, 66F/339L/410K/543Q, 66F/339L/474E, 66F/370E,66F/410K/424C/454V/527H, 66F/424C, 66F/463A/464C, 66F/463L/464Q,66F/543Q, 102S, 104G, 105G, 154T, 214Q/244S/543Q, 214Q/374D/424C, 227F,227F/244S/411A/424C, 227F/339L/413A/437G, 244S/411A, 339L, 394L,410K/411A/424G, 410L, 410T, 410V, 410Y, 413T, 421Q, 424G, 424L, 517D,524I, 554L, and 554V, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 988. Insome additional embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 988, or a functional fragment thereof, andwherein the engineered phenylalanine ammonia lyase comprises at leastone substitution or substitution set selected from N36C, N36Q,R40C/Y66F/V227F, R40C/Y66F/V227F/A244S/M410K/A424C,R40C/Y66F/M410K/N474E, R40C/M410K/E411A/A424C, P47A, P47E, P47R, P47T,Y66F, Y66F/L214Q/H374D/M410K/N474E, Y66F/L214Q/A424C,Y66F/L214Q/N437G/N474E, Y66F/V227F, Y66F/V227F/A244S/A424C/A543Q,Y66F/V227F/A424G, Y66F/I339L, Y66F/I339L/M410K/A543Q, Y66F/I339L/N474E,Y66F/M370E, Y66F/M410K/A424C/I454V/R527H, Y66F/A424C, Y66F/T463A/L464C,Y66F/T463L/L464Q, Y66F/A543Q, M102S, A104G, I105G, G154T,L214Q/A244S/A543Q, L214Q/H374D/A424C, V227F, V227F/A244S/E411A/A424C,V227F/I339L/K413A/N437G, A244S/E411A, I339L, N394L, M410K/E411A/A424G,M410L, M410T, M410V, M410Y, K413T, T421Q, A424G, A424L, E517D, A524I,R554L, and R554V, wherein the amino acid positions of the polypeptidesequence are numbered with reference to SEQ ID NO: 988.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 1140, or a functional fragment thereof. In someembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 1140, or a functional fragment thereof, and wherein theengineered phenylalanine ammonia lyase comprises at least onesubstitution or substitution set at one or more positions selected from36/47/424/517/554, 47/214/413/524/563, 47/410/524, 47/524, 47/554, 214,214/424, 410, 410/517/554, 410/554, 424/517/554, 517/524/554, and 554,wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 1140. In some additionalembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQID NO: 1140, or a functional fragment thereof, and wherein theengineered phenylalanine ammonia lyase comprises at least onesubstitution or substitution set selected from 36Q/47R/424L/517D/554V,47E/214Q/413A/524I/563V, 47E/524I, 47T/410Y/524I, 47T/554L, 214Q,214Q/424L, 410L/554V, 410T/517D/554V, 410Y, 424L/517D/554V,517D/524I/554L, and 554L, wherein the amino acid positions of thepolypeptide sequence are numbered with reference to SEQ ID NO: 1140. Insome additional embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence having at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO: 1140, or a functional fragment thereof, andwherein the engineered phenylalanine ammonia lyase comprises at leastone substitution or substitution set selected fromN36Q/P47R/A424L/E517D/R554V, P47E/L214Q/K413A/A524I/L563V, P47E/A524I,P47T/K410Y/A524I, P47T/R554L, L214Q, L214Q/A424L, K410L/R554V,K410T/E517D/R554V, K410Y, A424L/E517D/R554V, E517D/A524I/R554L, andR554L, wherein the amino acid positions of the polypeptide sequence arenumbered with reference to SEQ ID NO: 1140.

In some embodiments, the engineered phenylalanine ammonia lyasecomprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto SEQ ID NO: 2, 4, 8, 106, 252, 446, 482, 516, 618, 714, 830, 894, 988,and/or 1140, or a functional fragment thereof. In some additionalembodiments, the engineered phenylalanine ammonia lyase comprises SEQ IDNO: 4, 8, 106, 252, 446, 482, 516, 618, 714, 830, 894, 988, or 1140, ora functional fragment thereof. In still some additional embodiments, theengineered phenylalanine ammonia lyase comprises a polypeptide sequencethat is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the sequence of at least oneengineered phenylalanine ammonia lyase variant set forth in Table 5.1,6.1, 7.1, 8.1, 9.1, 10.1, 11.1. 12.1, 13.1, 14.1, 15.1, 18.1, and/or19.1. In yet some further embodiments, the engineered phenylalanineammonia lyase comprises a polypeptide sequence that is at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the sequence of at least one engineered phenylalanineammonia lyase variant set forth in the even numbered sequences of SEQ IDNOS: 4-1222. In some embodiments, the engineered phenylalanine ammonialyase comprises a polypeptide sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence ofat least one engineered phenylalanine ammonia lyase variant set forth inthe even numbered sequences of SEQ ID NOS: 4-1222. In some furtherembodiments, the engineered phenylalanine ammonia lyase comprises apolypeptide sequence set forth in the even numbered sequences of SEQ IDNOS: 4-1222. In some additional embodiments, the engineeredphenylalanine ammonia lyase comprises a polypeptide sequence exhibits atleast one improved property compared to wild-type Anabaena variabilisphenylalanine ammonia lyase. In some additional embodiments, theimproved property comprises improved production of compound 2. In someembodiments, the improved property comprises improved production of

In some additional embodiments, the improved property comprises improvedutilization of

In yet some additional embodiments, the improved property comprisesimproved production of

In yet some additional embodiments, the improved property comprisesimproved enantioselectivity. In some further embodiments, the improvedproperty comprises improved stability. In some additional embodiments,the improved property comprises improved thermostability, improved acidstability, and/or improved alkaline stability. In some furtherembodiments, the engineered phenylalanine ammonia lyase is purified. Inpresent invention also provides compositions comprising at least oneengineered phenylalanine ammonia lyase provided herein. In presentinvention also provides compositions comprising an engineeredphenylalanine ammonia lyase provided herein.

The present invention also provides engineered polynucleotide sequencesencoding at least one engineered phenylalanine ammonia lyase providedherein. In some embodiments, the engineered polynucleotide sequencecomprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 1, 3,7, 105, 251, 445, 481, 515, 617, 713, 829, 893, 987, and/or 1139. Insome additional embodiments, the engineered polynucleotide sequencecomprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 1, 3,7, 105, 251, 445, 481, 515, 617, 713, 829, 893, 987, and/or 1139,wherein the polynucleotide sequence of the engineered phenylalanineammonia lyase comprises at least one substitution at one or morepositions. In some additional embodiments, the engineered polynucleotidesequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 1,3, 7, 105, 251, 445, 481, 515, 617, 713, 829, 893, 987, and/or 1139. Insome embodiments, the engineered polynucleotide sequence comprises SEQID NO: 1, 3, 7, 105, 251, 445, 481, 515, 617, 713, 829, 893, 987, and/or1139. In some additional embodiments, the engineered polynucleotidesequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to one of theodd-numbered sequences in SEQ ID NO: 3-1221. In some furtherembodiments, the engineered polynucleotide sequence comprises a sequenceset forth in the odd-numbered sequences set forth in SEQ ID NOS: 3-1221.In some additional embodiments, the engineered polynucleotide sequenceis operably linked to a control sequence. In some embodiments, thecontrol sequence comprises a promoter. In some additional embodiments,the promoter is a heterologous promoter. In some further embodiments,the control sequence comprises more than one control sequence. In someembodiments, one of the control sequences comprises a promoter and theother control sequence comprises a different control sequence. In someembodiments, the engineered polynucleotide sequence is codon optimized.The present invention also provides expression vectors comprising atleast one polynucleotide sequence provided herein. The present inventionalso provides host cells comprising at least one expression vectorprovided herein. In some embodiments, the present invention provideshost cells comprising at least one polynucleotide sequence providedherein. In some embodiments, the host cell is a prokaryotic cell, whilein some other embodiments, the host cell is a eukaryotic cell. In someadditional embodiments, the host cell is E. coli.

The present invention also provides methods of producing an engineeredphenylalanine ammonia lyase in a host cell, comprising culturing thehost cell provided herein, in a culture medium under suitableconditions, such that at least one engineered phenylalanine ammonialyase provided herein is produced. In some embodiments, the methodsfurther comprise the step of recovering at least one engineeredphenylalanine ammonia lyase from the culture medium and/or the hostcell. In some additional embodiments, the methods further comprise thestep of purifying the at least one phenylalanine ammonia lyase obtainedusing the methods of the present invention.

DESCRIPTION OF THE INVENTION

The present invention provides engineered phenylalanine ammonia lyase(PAL) polypeptides and compositions thereof, as well as polynucleotidesencoding the engineered phenylalanine ammonia lyase (PAL) polypeptides.Methods for producing PAL enzymes are also provided. In someembodiments, the engineered PAL polypeptides are optimized to provideenhanced catalytic activities that are useful under industrial processconditions for the production of pharmaceutical compounds.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains. Generally,the nomenclature used herein and the laboratory procedures of cellculture, molecular genetics, microbiology, organic chemistry, analyticalchemistry and nucleic acid chemistry described below are thosewell-known and commonly employed in the art. Such techniques arewell-known and described in numerous texts and reference works wellknown to those of skill in the art. Standard techniques, ormodifications thereof, are used for chemical syntheses and chemicalanalyses. Accordingly, the following terms are intended to have themeanings provided herein.

Although any suitable methods and materials similar or equivalent tothose described herein find use in the practice of the presentinvention, some methods and materials are described herein. It is to beunderstood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context they are used by those of skill in the art.Accordingly, the terms defined immediately below are more fullydescribed by reference to the invention as a whole.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the present invention. The section headingsused herein are for organizational purposes only and not to be construedas limiting the subject matter described. Numeric ranges are inclusiveof the numbers defining the range. Thus, every numerical range disclosedherein is intended to encompass every narrower numerical range thatfalls within such broader numerical range, as if such narrower numericalranges were all expressly written herein. It is also intended that everymaximum (or minimum) numerical limitation disclosed herein includesevery lower (or higher) numerical limitation, as if such lower (orhigher) numerical limitations were expressly written herein.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a polypeptide” includes more than onepolypeptide.

Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,”and “including” are interchangeable and not intended to be limiting.Thus, as used herein, the term “comprising” and its cognates are used intheir inclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

All patents, patent applications, articles and publications mentionedherein, both supra and infra, are hereby expressly incorporated hereinby reference.

Definitions and Abbreviations

The abbreviations used for the genetically encoded amino acids areconventional and are as follows: alanine (Ala or A), arginine (Arg orR), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C),glutamate (Glu or E), glutamine (Gln or Q), histidine (His or H),isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine(Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser orS), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y),and valine (Val or V).

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the amino (N) to carboxy (C)direction in accordance with common convention.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleosides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When nucleic acid sequences are presented as astring of one-letter abbreviations, the sequences are presented in the5′ to 3′ direction in accordance with common convention, and thephosphates are not indicated.

As used herein, the term “about” means an acceptable error for aparticular value. In some instances “about” means within 0.05%, 0.5%,1.0%, or 2.0%, of a given value range. In some instances, “about” meanswithin 1, 2, 3, or 4 standard deviations of a given value.

As used herein, “EC” number refers to the Enzyme Nomenclature of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (NC-IUBMB). The IUBMB biochemical classification is anumerical classification system for enzymes based on the chemicalreactions they catalyze.

As used herein, “ATCC” refers to the American Type Culture Collectionwhose biorepository collection includes genes and strains.

As used herein, “NCBI” refers to National Center for BiologicalInformation and the sequence databases provided therein.

As used herein, “phenylalanine ammonia lysate” (“PAL”) enzymes areenzymes that catalyze the reversible non-oxidative deamination ofL-phenylalanine and related compounds such asL-2-amino-3-(2-(benzyloxy)-3-methoxyphenyl)propanoic acid.

“Protein,” “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation or phosphorylation). Included within thisdefinition are D- and L-amino acids, and mixtures of D- and L-aminoacids, as well as polymers comprising D- and L-amino acids, and mixturesof D- and L-amino acids.

“Amino acids” are referred to herein by either their commonly knownthree-letter symbols or by the one-letter symbols recommended byIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single letter codes.

As used herein, “hydrophilic amino acid or residue” refers to an aminoacid or residue having a side chain exhibiting a hydrophobicity of lessthan zero according to the normalized consensus hydrophobicity scale ofEisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142 [1984]).Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser(S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K)and L-Arg (R).

As used herein, “acidic amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain exhibiting a pKa value of lessthan about 6 when the amino acid is included in a peptide orpolypeptide. Acidic amino acids typically have negatively charged sidechains at physiological pH due to loss of a hydrogen ion. Geneticallyencoded acidic amino acids include L-Glu (E) and L-Asp (D).

As used herein, “basic amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain exhibiting a pKa value ofgreater than about 6 when the amino acid is included in a peptide orpolypeptide. Basic amino acids typically have positively charged sidechains at physiological pH due to association with hydronium ion.Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

As used herein, “polar amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain that is uncharged atphysiological pH, but which has at least one bond in which the pair ofelectrons shared in common by two atoms is held more closely by one ofthe atoms. Genetically encoded polar amino acids include L-Asn (N),L-Gln (Q), L-Ser (S) and L-Thr (T).

As used herein, “hydrophobic amino acid or residue” refers to an aminoacid or residue having a side chain exhibiting a hydrophobicity ofgreater than zero according to the normalized consensus hydrophobicityscale of Eisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142[1984]). Genetically encoded hydrophobic amino acids include L-Pro (P),L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala(A) and L-Tyr (Y).

As used herein, “aromatic amino acid or residue” refers to a hydrophilicor hydrophobic amino acid or residue having a side chain that includesat least one aromatic or heteroaromatic ring. Genetically encodedaromatic amino acids include L-Phe (F), L-Tyr (Y) and L-Trp (W).Although owing to the pKa of its heteroaromatic nitrogen atom L-His (H)it is sometimes classified as a basic residue, or as an aromatic residueas its side chain includes a heteroaromatic ring, herein histidine isclassified as a hydrophilic residue or as a “constrained residue” (seebelow).

As used herein, “constrained amino acid or residue” refers to an aminoacid or residue that has a constrained geometry. Herein, constrainedresidues include L-Pro (P) and L-His (H). Histidine has a constrainedgeometry because it has a relatively small imidazole ring. Proline has aconstrained geometry because it also has a five membered ring.

As used herein, “non-polar amino acid or residue” refers to ahydrophobic amino acid or residue having a side chain that is unchargedat physiological pH and which has bonds in which the pair of electronsshared in common by two atoms is generally held equally by each of thetwo atoms (i.e., the side chain is not polar). Genetically encodednon-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile(I), L-Met (M) and L-Ala (A).

As used herein, “aliphatic amino acid or residue” refers to ahydrophobic amino acid or residue having an aliphatic hydrocarbon sidechain. Genetically encoded aliphatic amino acids include L-Ala (A),L-Val (V), L-Leu (L) and L-Ile (I). It is noted that cysteine (or“L-Cys” or “[C]”) is unusual in that it can form disulfide bridges withother L-Cys (C) amino acids or other sulfanyl- or sulfhydryl-containingamino acids. The “cysteine-like residues” include cysteine and otheramino acids that contain sulfhydryl moieties that are available forformation of disulfide bridges. The ability of L-Cys (C) (and otheramino acids with —SH containing side chains) to exist in a peptide ineither the reduced free —SH or oxidized disulfide-bridged form affectswhether L-Cys (C) contributes net hydrophobic or hydrophilic characterto a peptide. While L-Cys (C) exhibits a hydrophobicity of 0.29according to the normalized consensus scale of Eisenberg (Eisenberg etal., 1984, supra), it is to be understood that for purposes of thepresent disclosure, L-Cys (C) is categorized into its own unique group.

As used herein, “small amino acid or residue” refers to an amino acid orresidue having a side chain that is composed of a total three or fewercarbon and/or heteroatoms (excluding the α-carbon and hydrogens). Thesmall amino acids or residues may be further categorized as aliphatic,non-polar, polar or acidic small amino acids or residues, in accordancewith the above definitions. Genetically-encoded small amino acidsinclude L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T)and L-Asp (D).

As used herein, “hydroxyl-containing amino acid or residue” refers to anamino acid containing a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr(Y).

As used herein, “polynucleotide” and “nucleic acid” refer to two or morenucleotides that are covalently linked together. The polynucleotide maybe wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of2′-deoxyribonucleotides (i.e., DNA), or comprised of mixtures of ribo-and 2′-deoxyribonucleotides. While the nucleosides will typically belinked together via standard phosphodiester linkages, thepolynucleotides may include one or more non-standard linkages. Thepolynucleotide may be single-stranded or double-stranded, or may includeboth single-stranded regions and double-stranded regions. Moreover,while a polynucleotide will typically be composed of the naturallyoccurring encoding nucleobases (i.e., adenine, guanine, uracil, thymineand cytosine), it may include one or more modified and/or syntheticnucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc.In some embodiments, such modified or synthetic nucleobases arenucleobases encoding amino acid sequences.

As used herein, “nucleoside” refers to glycosylamines comprising anucleobase (i.e., a nitrogenous base), and a 5-carbon sugar (e.g.,ribose or deoxyribose). Non-limiting examples of nucleosides includecytidine, uridine, adenosine, guanosine, thymidine, and inosine. Incontrast, the term “nucleotide” refers to the glycosylamines comprisinga nucleobase, a 5-carbon sugar, and one or more phosphate groups. Insome embodiments, nucleosides can be phosphorylated by kinases toproduce nucleotides.

As used herein, “nucleoside diphosphate” refers to glycosylaminescomprising a nucleobase (i.e., a nitrogenous base), a 5-carbon sugar(e.g., ribose or deoxyribose), and a diphosphate (i.e., pyrophosphate)moiety. In some embodiments herein, “nucleoside diphosphate” isabbreviated as “NDP.” Non-limiting examples of nucleoside diphosphatesinclude cytidine diphosphate (CDP), uridine diphosphate (UDP), adenosinediphosphate (ADP), guanosine diphosphate (GDP), thymidine diphosphate(TDP), and inosine diphosphate. The terms “nucleoside” and “nucleotide”may be used interchangeably in some contexts.

As used herein, “coding sequence” refers to that portion of a nucleicacid (e.g., a gene) that encodes an amino acid sequence of a protein.

As used herein, the terms “biocatalysis,” “biocatalytic,”“biotransformation,” and “biosynthesis” refer to the use of enzymes toperform chemical reactions on organic compounds.

As used herein, “wild-type” and “naturally-occurring” refer to the formfound in nature. For example a wild-type polypeptide or polynucleotidesequence is a sequence present in an organism that can be isolated froma source in nature and which has not been intentionally modified byhuman manipulation.

As used herein, “recombinant,” “engineered,” “non-naturally occurring,”and “variant,” when used with reference to a cell, nucleic acid, orpolypeptide, refers to a material, or a material corresponding to thenatural or native form of the material, that has been modified in amanner that would not otherwise exist in nature. In some embodiments,the cell, nucleic acid or polypeptide is identical a naturally occurringcell, nucleic acid or polypeptide, but is produced or derived fromsynthetic materials and/or by manipulation using recombinant techniques.Non-limiting examples include, among others, recombinant cellsexpressing genes that are not found within the native (non-recombinant)form of the cell or express native genes that are otherwise expressed ata different level.

The term “percent (%) sequence identity” is used herein to refer tocomparisons among polynucleotides or polypeptides, and are determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage may be calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Alternatively, thepercentage may be calculated by determining the number of positions atwhich either the identical nucleic acid base or amino acid residueoccurs in both sequences or a nucleic acid base or amino acid residue isaligned with a gap to yield the number of matched positions, dividingthe number of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Those of skill in the art appreciatethat there are many established algorithms available to align twosequences. Optimal alignment of sequences for comparison can beconducted by any suitable method, including, but not limited to thelocal homology algorithm of Smith and Waterman (Smith and Waterman, Adv.Appl. Math., 2:482 [1981]), by the homology alignment algorithm ofNeedleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443[1970]), by the search for similarity method of Pearson and Lipman(Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]), bycomputerized implementations of these algorithms (e.g., GAP, BESTFIT,FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visualinspection, as known in the art. Examples of algorithms that aresuitable for determining percent sequence identity and sequencesimilarity include, but are not limited to the BLAST and BLAST 2.0algorithms, which are described by Altschul et al. (See Altschul et al.,J. Mol. Biol., 215: 403-410 [1990]; and Altschul et al., Nucl. AcidsRes., 3389-3402 [1977], respectively). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (See, Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix(See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915[1989]). Exemplary determination of sequence alignment and % sequenceidentity can employ the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.), using default parametersprovided.

As used herein, “reference sequence” refers to a defined sequence usedas a basis for a sequence and/or activity comparison. A referencesequence may be a subset of a larger sequence, for example, a segment ofa full-length gene or polypeptide sequence. Generally, a referencesequence is at least 20 nucleotide or amino acid residues in length, atleast 25 residues in length, at least 50 residues in length, at least100 residues in length or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptides aretypically performed by comparing sequences of the two polynucleotides orpolypeptides over a “comparison window” to identify and compare localregions of sequence similarity. In some embodiments, a “referencesequence” can be based on a primary amino acid sequence, where thereference sequence is a sequence that can have one or more changes inthe primary sequence.

As used herein, “comparison window” refers to a conceptual segment of atleast about 20 contiguous nucleotide positions or amino acid residueswherein a sequence may be compared to a reference sequence of at least20 contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

As used herein, “corresponding to,” “reference to,” and “relative to”when used in the context of the numbering of a given amino acid orpolynucleotide sequence refer to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence. In other words, theresidue number or residue position of a given polymer is designated withrespect to the reference sequence rather than by the actual numericalposition of the residue within the given amino acid or polynucleotidesequence. For example, a given amino acid sequence, such as that of anengineered phenylalanine ammonia lyase, can be aligned to a referencesequence by introducing gaps to optimize residue matches between the twosequences. In these cases, although the gaps are present, the numberingof the residue in the given amino acid or polynucleotide sequence ismade with respect to the reference sequence to which it has beenaligned.

As used herein, “substantial identity” refers to a polynucleotide orpolypeptide sequence that has at least 80 percent sequence identity, atleast 85 percent identity, at least between 89 to 95 percent sequenceidentity, or more usually, at least 99 percent sequence identity ascompared to a reference sequence over a comparison window of at least 20residue positions, frequently over a window of at least 30-50 residues,wherein the percentage of sequence identity is calculated by comparingthe reference sequence to a sequence that includes deletions oradditions which total 20 percent or less of the reference sequence overthe window of comparison. In some specific embodiments applied topolypeptides, the term “substantial identity” means that two polypeptidesequences, when optimally aligned, such as by the programs GAP orBESTFIT using default gap weights, share at least 80 percent sequenceidentity, preferably at least 89 percent sequence identity, at least 95percent sequence identity or more (e.g., 99 percent sequence identity).In some embodiments, residue positions that are not identical insequences being compared differ by conservative amino acidsubstitutions.

As used herein, “amino acid difference” and “residue difference” referto a difference in the amino acid residue at a position of a polypeptidesequence relative to the amino acid residue at a corresponding positionin a reference sequence. In some cases, the reference sequence has ahistidine tag, but the numbering is maintained relative to theequivalent reference sequence without the histidine tag. The positionsof amino acid differences generally are referred to herein as “Xn,”where n refers to the corresponding position in the reference sequenceupon which the residue difference is based. For example, a “residuedifference at position X93 as compared to SEQ ID NO:4” refers to adifference of the amino acid residue at the polypeptide positioncorresponding to position 93 of SEQ ID NO:4. Thus, if the referencepolypeptide of SEQ ID NO:4 has a serine at position 93, then a “residuedifference at position X93 as compared to SEQ ID NO:4” an amino acidsubstitution of any residue other than serine at the position of thepolypeptide corresponding to position 93 of SEQ ID NO:4. In mostinstances herein, the specific amino acid residue difference at aposition is indicated as “XnY” where “Xn” specified the correspondingposition as described above, and “Y” is the single letter identifier ofthe amino acid found in the engineered polypeptide (i.e., the differentresidue than in the reference polypeptide). In some instances (e.g., inthe Tables presented in the Examples), the present invention alsoprovides specific amino acid differences denoted by the conventionalnotation “AnB”, where A is the single letter identifier of the residuein the reference sequence, “n” is the number of the residue position inthe reference sequence, and B is the single letter identifier of theresidue substitution in the sequence of the engineered polypeptide. Insome instances, a polypeptide of the present invention can include oneor more amino acid residue differences relative to a reference sequence,which is indicated by a list of the specified positions where residuedifferences are present relative to the reference sequence. In someembodiments, where more than one amino acid can be used in a specificresidue position of a polypeptide, the various amino acid residues thatcan be used are separated by a “/” (e.g., X307H/X307P or X307H/P). Theslash may also be used to indicate multiple substitutions within a givenvariant (i.e., there is more than one substitution present in a givensequence, such as in a combinatorial variant). In some embodiments, thepresent invention includes engineered polypeptide sequences comprisingone or more amino acid differences comprising conservative ornon-conservative amino acid substitutions. In some additionalembodiments, the present invention provides engineered polypeptidesequences comprising both conservative and non-conservative amino acidsubstitutions.

As used herein, “conservative amino acid substitution” refers to asubstitution of a residue with a different residue having a similar sidechain, and thus typically involves substitution of the amino acid in thepolypeptide with amino acids within the same or similar defined class ofamino acids. By way of example and not limitation, in some embodiments,an amino acid with an aliphatic side chain is substituted with anotheraliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine);an amino acid with an hydroxyl side chain is substituted with anotheramino acid with an hydroxyl side chain (e.g., serine and threonine); anamino acids having aromatic side chains is substituted with anotheramino acid having an aromatic side chain (e.g., phenylalanine, tyrosine,tryptophan, and histidine); an amino acid with a basic side chain issubstituted with another amino acid with a basis side chain (e.g.,lysine and arginine); an amino acid with an acidic side chain issubstituted with another amino acid with an acidic side chain (e.g.,aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilicamino acid is replaced with another hydrophobic or hydrophilic aminoacid, respectively.

As used herein, “non-conservative substitution” refers to substitutionof an amino acid in the polypeptide with an amino acid withsignificantly differing side chain properties. Non-conservativesubstitutions may use amino acids between, rather than within, thedefined groups and affects (a) the structure of the peptide backbone inthe area of the substitution (e.g., proline for glycine) (b) the chargeor hydrophobicity, or (c) the bulk of the side chain. By way of exampleand not limitation, an exemplary non-conservative substitution can be anacidic amino acid substituted with a basic or aliphatic amino acid; anaromatic amino acid substituted with a small amino acid; and ahydrophilic amino acid substituted with a hydrophobic amino acid.

As used herein, “deletion” refers to modification to the polypeptide byremoval of one or more amino acids from the reference polypeptide.Deletions can comprise removal of 1 or more amino acids, 2 or more aminoacids, 5 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, or up to 20% of the total number of amino acids making up thereference enzyme while retaining enzymatic activity and/or retaining theimproved properties of an engineered phenylalanine ammonia lyase enzyme.Deletions can be directed to the internal portions and/or terminalportions of the polypeptide. In various embodiments, the deletion cancomprise a continuous segment or can be discontinuous. Deletions aretypically indicated by “−” in amino acid sequences.

As used herein, “insertion” refers to modification to the polypeptide byaddition of one or more amino acids from the reference polypeptide.Insertions can be in the internal portions of the polypeptide, or to thecarboxy or amino terminus. Insertions as used herein include fusionproteins as is known in the art. The insertion can be a contiguoussegment of amino acids or separated by one or more of the amino acids inthe naturally occurring polypeptide.

The term “amino acid substitution set” or “substitution set” refers to agroup of amino acid substitutions in a polypeptide sequence, as comparedto a reference sequence. A substitution set can have 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. Insome embodiments, a substitution set refers to the set of amino acidsubstitutions that is present in any of the variant phenylalanineammonia lyases listed in the Tables provided in the Examples.

A “functional fragment” and “biologically active fragment” are usedinterchangeably herein to refer to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion(s) and/or internaldeletions, but where the remaining amino acid sequence is identical tothe corresponding positions in the sequence to which it is beingcompared (e.g., a full-length engineered phenylalanine ammonia lyase ofthe present invention) and that retains substantially all of theactivity of the full-length polypeptide.

As used herein, “isolated polypeptide” refers to a polypeptide which issubstantially separated from other contaminants that naturally accompanyit (e.g., protein, lipids, and polynucleotides). The term embracespolypeptides which have been removed or purified from theirnaturally-occurring environment or expression system (e.g., within ahost cell or via in vitro synthesis). The recombinant phenylalanineammonia lyase polypeptides may be present within a cell, present in thecellular medium, or prepared in various forms, such as lysates orisolated preparations. As such, in some embodiments, the recombinantphenylalanine ammonia lyase polypeptides can be an isolated polypeptide.

As used herein, “substantially pure polypeptide” or “purified protein”refers to a composition in which the polypeptide species is thepredominant species present (i.e., on a molar or weight basis it is moreabundant than any other individual macromolecular species in thecomposition), and is generally a substantially purified composition whenthe object species comprises at least about 50 percent of themacromolecular species present by mole or % weight. However, in someembodiments, the composition comprising phenylalanine ammonia lyasecomprises phenylalanine ammonia lyase that is less than 50% pure (e.g.,about 10%, about 20%, about 30%, about 40%, or about 50%) Generally, asubstantially pure phenylalanine ammonia lyase composition comprisesabout 60% or more, about 70% or more, about 80% or more, about 90% ormore, about 95% or more, and about 98% or more of all macromolecularspecies by mole or % weight present in the composition. In someembodiments, the object species is purified to essential homogeneity(i.e., contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species. Solvent species, smallmolecules (<500 Daltons), and elemental ion species are not consideredmacromolecular species. In some embodiments, the isolated recombinantphenylalanine ammonia lyase polypeptides are substantially purepolypeptide compositions.

As used herein, “improved enzyme property” refers to at least oneimproved property of an enzyme. In some embodiments, the presentinvention provides engineered phenylalanine ammonia lyase polypeptidesthat exhibit an improvement in any enzyme property as compared to areference phenylalanine ammonia lyase polypeptide and/or a wild-typephenylalanine ammonia lyase polypeptide, and/or another engineeredphenylalanine ammonia lyase polypeptide. Thus, the level of“improvement” can be determined and compared between variousphenylalanine ammonia lyase polypeptides, including wild-type, as wellas engineered phenylalanine ammonia lyase. Improved properties include,but are not limited, to such properties as increased protein expression,increased thermoactivity, increased thermostability, increased pHactivity, increased stability, increased enzymatic activity, increasedsubstrate specificity or affinity, increased specific activity,increased resistance to substrate or end-product inhibition, increasedchemical stability, improved chemoselectivity, improved solventstability, increased tolerance to acidic pH, increased tolerance toproteolytic activity (i.e., reduced sensitivity to proteolysis), reducedaggregation, increased solubility, and altered temperature profile. Inadditional embodiments, the term is used in reference to the at leastone improved property of phenylalanine ammonia lyase enzymes. In someembodiments, the present invention provides engineered phenylalanineammonia lyase polypeptides that exhibit an improvement in any enzymeproperty as compared to a reference phenylalanine ammonia lyasepolypeptide and/or a wild-type phenylalanine ammonia lyase polypeptide,and/or another engineered phenylalanine ammonia lyase polypeptide. Thus,the level of “improvement” can be determined and compared betweenvarious phenylalanine ammonia lyase polypeptides, including wild-type,as well as engineered phenylalanine ammonia lyases.

As used herein, “increased enzymatic activity” and “enhanced catalyticactivity” refer to an improved property of the engineered polypeptides,which can be represented by an increase in specific activity (e.g.,product produced/time/weight protein) or an increase in percentconversion of the substrate to the product (e.g., percent conversion ofstarting amount of substrate to product in a specified time period usinga specified amount of enzyme) as compared to the reference enzyme. Insome embodiments, the terms refer to an improved property of engineeredphenylalanine ammonia lyase polypeptides provided herein, which can berepresented by an increase in specific activity (e.g., productproduced/time/weight protein) or an increase in percent conversion ofthe substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of phenylalanine ammonia lyase) as compared to thereference phenylalanine ammonia lyase enzyme. In some embodiments, theterms are used in reference to improved phenylalanine ammonia lyaseenzymes provided herein. Exemplary methods to determine enzyme activityof the engineered phenylalanine ammonia lyases of the present inventionare provided in the Examples. Any property relating to enzyme activitymay be affected, including the classical enzyme properties of K_(m),V_(max) or k_(cat), changes of which can lead to increased enzymaticactivity. For example, improvements in enzyme activity can be from about1.1 fold the enzymatic activity of the corresponding wild-type enzyme,to as much as 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold,75-fold, 100-fold, 150-fold, 200-fold or more enzymatic activity thanthe naturally occurring phenylalanine ammonia lyase or anotherengineered phenylalanine ammonia lyase from which the phenylalanineammonia lyase polypeptides were derived.

As used herein, “conversion” refers to the enzymatic conversion (orbiotransformation) of a substrate(s) to the corresponding product(s).“Percent conversion” refers to the percent of the substrate that isconverted to the product within a period of time under specifiedconditions. Thus, the “enzymatic activity” or “activity” of aphenylalanine ammonia lyase polypeptide can be expressed as “percentconversion” of the substrate to the product in a specific period oftime.

Enzymes with “generalist properties” (or “generalist enzymes”) refer toenzymes that exhibit improved activity for a wide range of substrates,as compared to a parental sequence. Generalist enzymes do notnecessarily demonstrate improved activity for every possible substrate.In some embodiments, the present invention provides phenylalanineammonia lyase variants with generalist properties, in that theydemonstrate similar or improved activity relative to the parental genefor a wide range of sterically and electronically diverse substrates. Inaddition, the generalist enzymes provided herein were engineered to beimproved across a wide range of diverse molecules to increase theproduction of metabolites/products.

The term “stringent hybridization conditions” is used herein to refer toconditions under which nucleic acid hybrids are stable. As known tothose of skill in the art, the stability of hybrids is reflected in themelting temperature (T_(m)) of the hybrids. In general, the stability ofa hybrid is a function of ion strength, temperature, G/C content, andthe presence of chaotropic agents. The T_(m) values for polynucleotidescan be calculated using known methods for predicting meltingtemperatures (See e.g., Baldino et al., Meth. Enzymol., 168:761-777[1989]; Bolton et al., Proc. Natl. Acad. Sci. USA 48:1390 [1962];Bresslauer et al., Proc. Natl. Acad. Sci. USA 83:8893-8897 [1986];Freier et al., Proc. Natl. Acad. Sci. USA 83:9373-9377 [1986]; Kierzeket al., Biochem., 25:7840-7846 [1986]; Rychlik et al., Nucl. Acids Res.,18:6409-6412 [1990] (erratum, Nucl. Acids Res., 19:698 [1991]); Sambrooket al., supra); Suggs et al., 1981, in Developmental Biology UsingPurified Genes, Brown et al. [eds.], pp. 683-693, Academic Press,Cambridge, Mass. [1981]; and Wetmur, Crit. Rev. Biochem. Mol. Biol.26:227-259 [1991]). In some embodiments, the polynucleotide encodes thepolypeptide disclosed herein and hybridizes under defined conditions,such as moderately stringent or highly stringent conditions, to thecomplement of a sequence encoding an engineered phenylalanine ammonialyase enzyme of the present invention.

As used herein, “hybridization stringency” relates to hybridizationconditions, such as washing conditions, in the hybridization of nucleicacids. Generally, hybridization reactions are performed under conditionsof lower stringency, followed by washes of varying but higherstringency. The term “moderately stringent hybridization” refers toconditions that permit target-DNA to bind a complementary nucleic acidthat has about 60% identity, preferably about 75% identity, about 85%identity to the target DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w/v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

As used herein, “codon optimized” refers to changes in the codons of thepolynucleotide encoding a protein to those preferentially used in aparticular organism such that the encoded protein is efficientlyexpressed in the organism of interest. Although the genetic code isdegenerate in that most amino acids are represented by several codons,called “synonyms” or “synonymous” codons, it is well known that codonusage by particular organisms is nonrandom and biased towards particularcodon triplets. This codon usage bias may be higher in reference to agiven gene, genes of common function or ancestral origin, highlyexpressed proteins versus low copy number proteins, and the aggregateprotein coding regions of an organism's genome. In some embodiments, thepolynucleotides encoding the glycosyltransferase enzymes may be codonoptimized for optimal production in the host organism selected forexpression.

As used herein, “preferred,” “optimal,” and “high codon usage bias”codons when used alone or in combination refer(s) interchangeably tocodons that are used at higher frequency in the protein coding regionsthan other codons that code for the same amino acid. The preferredcodons may be determined in relation to codon usage in a single gene, aset of genes of common function or origin, highly expressed genes, thecodon frequency in the aggregate protein coding regions of the wholeorganism, codon frequency in the aggregate protein coding regions ofrelated organisms, or combinations thereof. Codons whose frequencyincreases with the level of gene expression are typically optimal codonsfor expression. A variety of methods are known for determining the codonfrequency (e.g., codon usage, relative synonymous codon usage) and codonpreference in specific organisms, including multivariate analysis, forexample, using cluster analysis or correspondence analysis, and theeffective number of codons used in a gene (See e.g., GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, Peden,University of Nottingham; McInerney, Bioinform., 14:372-73 [1998];Stenico et al., Nucl. Acids Res., 222437-46 [1994]; and Wright, Gene87:23-29 [1990]). Codon usage tables are available for many differentorganisms (See e.g., Wada et al., Nucl. Acids Res., 20:2111-2118 [1992];Nakamura et al., Nucl. Acids Res., 28:292 [2000]; Duret, et al., supra;Henaut and Danchin, in Escherichia coli and Salmonella, Neidhardt, etal. (eds.), ASM Press, Washington D.C., p. 2047-2066 [1996]). The datasource for obtaining codon usage may rely on any available nucleotidesequence capable of coding for a protein. These data sets includenucleic acid sequences actually known to encode expressed proteins(e.g., complete protein coding sequences-CDS), expressed sequence tags(ESTS), or predicted coding regions of genomic sequences (See e.g.,Mount, Bioinformatics: Sequence and Genome Analysis, Chapter 8, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [2001];Uberbacher, Meth. Enzymol., 266:259-281 [1996]; and Tiwari et al.,Comput. Appl. Biosci., 13:263-270 [1997]).

As used herein, “control sequence” includes all components, which arenecessary or advantageous for the expression of a polynucleotide and/orpolypeptide of the present invention. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter sequence, signalpeptide sequence, initiation sequence and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest, such as acoding sequence. The promoter sequence contains transcriptional controlsequences, which mediate the expression of a polynucleotide of interest.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

The phrase “suitable reaction conditions” refers to those conditions inthe enzymatic conversion reaction solution (e.g., ranges of enzymeloading, substrate loading, temperature, pH, buffers, co-solvents, etc.)under which a phenylalanine ammonia lyase polypeptide of the presentinvention is capable of converting a substrate to the desired productcompound. Some exemplary “suitable reaction conditions” are providedherein.

As used herein, “loading,” such as in “compound loading” or “enzymeloading” refers to the concentration or amount of a component in areaction mixture at the start of the reaction.

As used herein, “substrate” in the context of an enzymatic conversionreaction process refers to the compound or molecule acted on by theengineered enzymes provided herein (e.g., engineered phenylalanineammonia lyase polypeptides).

As used herein, “increasing” yield of a product (e.g., anL-phenylalanine analog) from a reaction occurs when a particularcomponent present during the reaction (e.g., a phenylalanine ammonialyase enzyme) causes more product to be produced, compared with areaction conducted under the same conditions with the same substrate andother substituents, but in the absence of the component of interest.

A reaction is said to be “substantially free” of a particular enzyme ifthe amount of that enzyme compared with other enzymes that participatein catalyzing the reaction is less than about 2%, about 1%, or about0.1% (wt/wt).

As used herein, “fractionating” a liquid (e.g., a culture broth) meansapplying a separation process (e.g., salt precipitation, columnchromatography, size exclusion, and filtration) or a combination of suchprocesses to provide a solution in which a desired protein comprises agreater percentage of total protein in the solution than in the initialliquid product.

As used herein, “starting composition” refers to any composition thatcomprises at least one substrate. In some embodiments, the startingcomposition comprises any suitable substrate.

As used herein, “product” in the context of an enzymatic conversionprocess refers to the compound or molecule resulting from the action ofan enzymatic polypeptide on a substrate.

As used herein, “equilibration” as used herein refers to the processresulting in a steady state concentration of chemical species in achemical or enzymatic reaction (e.g., interconversion of two species Aand B), including interconversion of stereoisomers, as determined by theforward rate constant and the reverse rate constant of the chemical orenzymatic reaction.

As used herein, “alkyl” refers to saturated hydrocarbon groups of from 1to 18 carbon atoms inclusively, either straight chained or branched,more preferably from 1 to 8 carbon atoms inclusively, and mostpreferably 1 to 6 carbon atoms inclusively. An alkyl with a specifiednumber of carbon atoms is denoted in parenthesis (e.g., (C₁-C₄)alkylrefers to an alkyl of 1 to 4 carbon atoms).

As used herein, “alkenyl” refers to groups of from 2 to 12 carbon atomsinclusively, either straight or branched containing at least one doublebond but optionally containing more than one double bond.

As used herein, “alkynyl” refers to groups of from 2 to 12 carbon atomsinclusively, either straight or branched containing at least one triplebond but optionally containing more than one triple bond, andadditionally optionally containing one or more double bonded moieties.

As used herein, “heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” referto alkyl, alkenyl and alkynyl as defined herein in which one or more ofthe carbon atoms are each independently replaced with the same ordifferent heteroatoms or heteroatomic groups. Heteroatoms and/orheteroatomic groups which can replace the carbon atoms include, but arenot limited to, —O—, —S—, —S—O—, —NR^(α)—, —PH—, —S(O)—, —S(O)2-, —S(O)NR^(α)—, —S(O)₂NR^(α)—, and the like, including combinations thereof,where each Ra is independently selected from hydrogen, alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

As used herein, “alkoxy” refers to the group —OR^(β) wherein R^(β) is analkyl group is as defined above including optionally substituted alkylgroups as also defined herein.

As used herein, “aryl” refers to an unsaturated aromatic carbocyclicgroup of from 6 to 12 carbon atoms inclusively having a single ring(e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl).Exemplary aryls include phenyl, pyridyl, naphthyl and the like.

As used herein, “amino” refers to the group —NH₂. Substituted aminorefers to the group —NHR^(δ), NR^(δ)R^(δ), and NR^(δ)R^(δ)R^(δ), whereeach R^(δ) is independently selected from substituted or unsubstitutedalkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl,heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, andthe like. Typical amino groups include, but are limited to,dimethylamino, diethylamino, trimethylammonium, triethylammonium,methylysulfonylamino, furanyl-oxy-sulfamino, and the like.

As used herein, “oxo” refers to ═O.

As used herein, “oxy” refers to a divalent group —O—, which may havevarious substituents to form different oxy groups, including ethers andesters.

As used herein, “carboxy” refers to —COOH.

As used herein, “carbonyl” refers to —C(O)—, which may have a variety ofsubstituents to form different carbonyl groups including acids, acidhalides, aldehydes, amides, esters, and ketones.

As used herein, “alkyloxycarbonyl” refers to —C(O)OR^(ε), where R^(ε) isan alkyl group as defined herein, which can be optionally substituted.

As used herein, “aminocarbonyl” refers to —C(O)NH₂. Substitutedaminocarbonyl refers to —C(O)NR^(δ)R^(δ), where the amino groupNR^(δ)R^(δ) is as defined herein.

As used herein, “halogen” and “halo” refer to fluoro, chloro, bromo andiodo.

As used herein, “hydroxy” refers to —OH.

As used herein, “cyano” refers to —CN.

As used herein, “heteroaryl” refers to an aromatic heterocyclic group offrom 1 to 10 carbon atoms inclusively and 1 to 4 heteroatoms inclusivelyselected from oxygen, nitrogen and sulfur within the ring. Suchheteroaryl groups can have a single ring (e.g., pyridyl or furyl) ormultiple condensed rings (e.g., indolizinyl or benzothienyl).

As used herein, “heteroarylalkyl” refers to an alkyl substituted with aheteroaryl (i.e., heteroaryl-alkyl-groups), preferably having from 1 to6 carbon atoms inclusively in the alkyl moiety and from 5 to 12 ringatoms inclusively in the heteroaryl moiety. Such heteroarylalkyl groupsare exemplified by pyridylmethyl and the like.

As used herein, “heteroarylalkenyl” refers to an alkenyl substitutedwith a heteroaryl (i.e., heteroaryl-alkenyl-groups), preferably havingfrom 2 to 6 carbon atoms inclusively in the alkenyl moiety and from 5 to12 ring atoms inclusively in the heteroaryl moiety.

As used herein, “heteroarylalkynyl” refers to an alkynyl substitutedwith a heteroaryl (i.e., heteroaryl-alkynyl-groups), preferably havingfrom 2 to 6 carbon atoms inclusively in the alkynyl moiety and from 5 to12 ring atoms inclusively in the heteroaryl moiety.

As used herein, “heterocycle,” “heterocyclic,” and interchangeably“heterocycloalkyl,” refer to a saturated or unsaturated group having asingle ring or multiple condensed rings, from 2 to 10 carbon ring atomsinclusively and from 1 to 4 hetero ring atoms inclusively selected fromnitrogen, sulfur or oxygen within the ring. Such heterocyclic groups canhave a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiplecondensed rings (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl).Examples of heterocycles include, but are not limited to, furan,thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine,pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine,indoline and the like.

As used herein, “membered ring” is meant to embrace any cyclicstructure. The number preceding the term “membered” denotes the numberof skeletal atoms that constitute the ring. Thus, for example,cyclohexyl, pyridine, pyran and thiopyran are 6-membered rings andcyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

Unless otherwise specified, positions occupied by hydrogen in theforegoing groups can be further substituted with substituentsexemplified by, but not limited to, hydroxy, oxo, nitro, methoxy,ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy,fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl,alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl,hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy,alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl,alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido,cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl,acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substitutedaryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl,heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl,substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl,morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; andpreferred heteroatoms are oxygen, nitrogen, and sulfur. It is understoodthat where open valences exist on these substituents they can be furthersubstituted with alkyl, cycloalkyl, aryl, heteroaryl, and/or heterocyclegroups, that where these open valences exist on carbon they can befurther substituted by halogen and by oxygen-, nitrogen-, orsulfur-bonded substituents, and where multiple such open valences exist,these groups can be joined to form a ring, either by direct formation ofa bond or by formation of bonds to a new heteroatom, preferably oxygen,nitrogen, or sulfur. It is further understood that the abovesubstitutions can be made provided that replacing the hydrogen with thesubstituent does not introduce unacceptable instability to the moleculesof the present invention, and is otherwise chemically reasonable.

As used herein the term “culturing” refers to the growing of apopulation of microbial cells under any suitable conditions (e.g., usinga liquid, gel or solid medium).

Recombinant polypeptides can be produced using any suitable methodsknown in the art. Genes encoding the wild-type polypeptide of interestcan be cloned in vectors, such as plasmids, and expressed in desiredhosts, such as E. coli, etc. Variants of recombinant polypeptides can begenerated by various methods known in the art. Indeed, there is a widevariety of different mutagenesis techniques well known to those skilledin the art. In addition, mutagenesis kits are also available from manycommercial molecular biology suppliers. Methods are available to makespecific substitutions at defined amino acids (site-directed), specificor random mutations in a localized region of the gene (regio-specific),or random mutagenesis over the entire gene (e.g., saturationmutagenesis). Numerous suitable methods are known to those in the art togenerate enzyme variants, including but not limited to site-directedmutagenesis of single-stranded DNA or double-stranded DNA using PCR,cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, andchemical saturation mutagenesis, or any other suitable method known inthe art. Mutagenesis and directed evolution methods can be readilyapplied to enzyme-encoding polynucleotides to generate variant librariesthat can be expressed, screened, and assayed. Any suitable mutagenesisand directed evolution methods find use in the present invention and arewell known in the art (See e.g., U.S. Pat. Nos. 5,605,793, 5,811,238,5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679,6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638,6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883,6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198,6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377,6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964,6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910,6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675,6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105,6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603, 6,518,065,6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467,6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072,6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882,6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297,7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477,7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464,7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030,7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499, 7,904,249,7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001,8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346,8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, andall related US, as well as PCT and non-US counterparts; Ling et al.,Anal. Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol.,57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botsteinet al., Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7[1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene,34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290[1999]; Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameriet al., Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol.,15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A.,94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319[1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad.Sci. USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966;WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336, all of whichare incorporated herein by reference).

In some embodiments, the enzyme clones obtained following mutagenesistreatment are screened by subjecting the enzyme preparations to adefined temperature (or other assay conditions) and measuring the amountof enzyme activity remaining after heat treatments or other suitableassay conditions. Clones containing a polynucleotide encoding apolypeptide are then isolated from the gene, sequenced to identify thenucleotide sequence changes (if any), and used to express the enzyme ina host cell. Measuring enzyme activity from the expression libraries canbe performed using any suitable method known in the art (e.g., standardbiochemistry techniques, such as HPLC analysis).

After the variants are produced, they can be screened for any desiredproperty (e.g., high or increased activity, or low or reduced activity,increased thermal activity, increased thermal stability, and/or acidicpH stability, etc.). In some embodiments, “recombinant phenylalanineammonia lyase polypeptides” (also referred to herein as “engineeredphenylalanine ammonia lyase polypeptides,” “variant phenylalanineammonia lyase enzymes,” “phenylalanine ammonia lyase variants,” and“phenylalanine ammonia lyase combinatorial variants”) find use. In someembodiments, “recombinant phenylalanine ammonia lyase polypeptides”(also referred to as “engineered phenylalanine ammonia lyasepolypeptides,” “variant phenylalanine ammonia lyase enzymes,”“phenylalanine ammonia lyase variants,” and “phenylalanine ammonia lyasecombinatorial variants”) find use.

As used herein, a “vector” is a DNA construct for introducing a DNAsequence into a cell. In some embodiments, the vector is an expressionvector that is operably linked to a suitable control sequence capable ofeffecting the expression in a suitable host of the polypeptide encodedin the DNA sequence. In some embodiments, an “expression vector” has apromoter sequence operably linked to the DNA sequence (e.g., transgene)to drive expression in a host cell, and in some embodiments, alsocomprises a transcription terminator sequence.

As used herein, the term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation, andpost-translational modification. In some embodiments, the term alsoencompasses secretion of the polypeptide from a cell.

As used herein, the term “produces” refers to the production of proteinsand/or other compounds by cells. It is intended that the term encompassany step involved in the production of polypeptides including, but notlimited to, transcription, post-transcriptional modification,translation, and post-translational modification. In some embodiments,the term also encompasses secretion of the polypeptide from a cell.

As used herein, an amino acid or nucleotide sequence (e.g., a promotersequence, signal peptide, terminator sequence, etc.) is “heterologous”to another sequence with which it is operably linked if the twosequences are not associated in nature. For example a “heterologouspolynucleotide” is any polynucleotide that is introduced into a hostcell by laboratory techniques, and includes polynucleotides that areremoved from a host cell, subjected to laboratory manipulation, and thenreintroduced into a host cell.

As used herein, the terms “host cell” and “host strain” refer tosuitable hosts for expression vectors comprising DNA provided herein(e.g., the polynucleotides encoding the phenylalanine ammonia lyasevariants). In some embodiments, the host cells are prokaryotic oreukaryotic cells that have been transformed or transfected with vectorsconstructed using recombinant DNA techniques as known in the art.

The term “analogue” means a polypeptide having more than 70% sequenceidentity but less than 100% sequence identity (e.g., more than 75%, 78%,80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity) with a reference polypeptide. In some embodiments,analogues means polypeptides that contain one or more non-naturallyoccurring amino acid residues including, but not limited, tohomoarginine, ornithine and norvaline, as well as naturally occurringamino acids. In some embodiments, analogues also include one or moreD-amino acid residues and non-peptide linkages between two or more aminoacid residues.

The term “effective amount” means an amount sufficient to produce thedesired result. One of general skill in the art may determine what theeffective amount by using routine experimentation.

The terms “isolated” and “purified” are used to refer to a molecule(e.g., an isolated nucleic acid, polypeptide, etc.) or other componentthat is removed from at least one other component with which it isnaturally associated. The term “purified” does not require absolutepurity, rather it is intended as a relative definition.

As used herein, “stereoselectivity” refers to the preferential formationin a chemical or enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (“e.e.”) calculated therefromaccording to the formula [major enantiomer—minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (“d.e.”).Enantiomeric excess and diastereomeric excess are types of stereomericexcess.

As used herein, “regioselectivity” and “regioselective reaction” referto a reaction in which one direction of bond making or breaking occurspreferentially over all other possible directions. Reactions cancompletely (100%) regioselective if the discrimination is complete,substantially regioselective (at least 75%), or partially regioselective(x %, wherein the percentage is set dependent upon the reaction ofinterest), if the product of reaction at one site predominates over theproduct of reaction at other sites.

As used herein, “chemoselectivity” refers to the preferential formationin a chemical or enzymatic reaction of one product over another.

As used herein, “pH stable” refers to a phenylalanine ammonia lyasepolypeptide that maintains similar activity (e.g., more than 60% to 80%)after exposure to high or low pH (e.g., 4.5-6 or 8 to 12) for a periodof time (e.g., 0.5-24 hrs) compared to the untreated enzyme.

As used herein, “thermostable” refers to a phenylalanine ammonia lyasepolypeptide that maintains similar activity (more than 60% to 80% forexample) after exposure to elevated temperatures (e.g., 40-80° C.) for aperiod of time (e.g., 0.5-24 h) compared to the wild-type enzyme exposedto the same elevated temperature.

As used herein, “solvent stable” refers to a phenylalanine ammonia lyasepolypeptide that maintains similar activity (more than e.g., 60% to 80%)after exposure to varying concentrations (e.g., 5-99%) of solvent(ethanol, isopropyl alcohol, dimethylsulfoxide [DMSO], tetrahydrofuran,2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyltert-butyl ether, etc.) for a period of time (e.g., 0.5-24 h) comparedto the wild-type enzyme exposed to the same concentration of the samesolvent.

As used herein, “thermo- and solvent stable” refers to a phenylalanineammonia lyase polypeptide that is both thermostable and solvent stable.

As used herein, “optional” and “optionally” mean that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. One of ordinary skill in the artwould understand that with respect to any molecule described ascontaining one or more optional substituents, only sterically practicaland/or synthetically feasible compounds are meant to be included.

As used herein, “optionally substituted” refers to all subsequentmodifiers in a term or series of chemical groups. For example, in theterm “optionally substituted arylalkyl, the “alkyl” portion and the“aryl” portion of the molecule may or may not be substituted, and forthe series “optionally substituted alkyl, cycloalkyl, aryl andheteroaryl,” the alkyl, cycloalkyl, aryl, and heteroaryl groups,independently of the others, may or may not be substituted.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides engineered phenylalanine ammonia lyase(PAL) polypeptides and compositions thereof, as well as polynucleotidesencoding the engineered phenylalanine ammonia lyase (PAL) polypeptides.Methods for producing PAL enzymes are also provided. In someembodiments, the engineered PAL polypeptides are optimized to provideenhanced catalytic activities that are useful under industrial processconditions for the production of pharmaceutical compounds.

In some embodiments, the present invention provides enzymes suitable forthe production of L-phenylalanine analogues such as EMA401-A1(Novartis). The present invention was developed in order to address thepotential use of enzymes to produce these L-phenylalanine analogues.However, it was determined that one challenge with this approach is thatwild-type enzymes are unlikely to be optimal for the required substrateanalogues needed for the production of L-phenylalanine analogues

Of particular interest is the development of PAL enzymes capable ofcatalyzing the reaction shown in Scheme 2. Compound (2), also known asEMA401-A1 is a precursor to compound (3), also known as EMA401, as shownin Scheme 3. EMA401 is first in class as a high-affinity ligand for theangiotensin II type 2 (AT2R) receptor and is being investigated for thetreatment of neuropathic pain (See, Hesselink and Schatman, J. PainRes., 10:439-443 [2017]). Prior to the development of the presentinvention, it was expected to be very difficult to identify naturallyoccurring PAL enzymes with sufficient activity on compound (1) forcommercial application, due to bulky nature of the substituents on thebenzyl ring (i.e., the benzyloxy and methoxy groups) and the electrondonating nature of these groups, as this has been described tonegatively influence PAL activity (See, Ahmed et al., ACS Catal.,8:3129-3132 [2018]). Thus, the present invention was developed in orderto address the need to engineer these enzymes for new or improvedactivity on compound (1), shown in Scheme 2, below.

The present invention provides engineered PAL polypeptides,polynucleotides encoding the polypeptides, methods of preparing thepolypeptides, and methods for using the polypeptides. Where thedescription relates to polypeptides, it is to be understood that it alsodescribes the polynucleotides encoding the polypeptides.

In some embodiments, the present invention provides engineered,non-naturally occurring PAL enzymes with improved properties as comparedto wild-type PAL enzymes. Any suitable reaction conditions find use inthe present invention. In some embodiments, methods are used to analyzethe improved properties of the engineered polypeptides to carry out theisomerization reaction. In some embodiments, the reaction conditions aremodified with regard to concentrations or amounts of engineered PAL,substrate(s), buffer(s), solvent(s), pH, conditions includingtemperature and reaction time, and/or conditions with the engineered PALpolypeptide immobilized on a solid support, as further described belowand in the Examples. In some embodiments, additional reaction componentsor additional techniques are utilized to supplement the reactionconditions. In some embodiments, these include taking measures tostabilize or prevent inactivation of the enzyme, reduce productinhibition, shift reaction equilibrium to desired product formation.

In some further embodiments, any of the above described processes forthe conversion of substrate compound to product compound can furthercomprise one or more steps selected from: extraction, isolation,purification, crystallization, filtration, and/or lyophilization ofproduct compound(s). Methods, techniques, and protocols for extracting,isolating, purifying, and/or crystallizing the product(s) frombiocatalytic reaction mixtures produced by the processes provided hereinare known to the ordinary artisan and/or accessed through routineexperimentation. Additionally, illustrative methods are provided in theExamples below.

Engineered PAL Polypeptides

In some additional embodiments, the engineered phenylalanine ammonialyase polypeptide of the present invention comprises a polypeptidecomprising at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 2, 4, 8, 106, 252, 446, 482, 516, 618,714, 830, 894, 988, and/or 1140.

In some embodiments, engineered phenylalanine ammonia lyase polypeptidesare produced by cultivating a microorganism comprising at least onepolynucleotide sequence encoding at least one engineered phenylalanineammonia lyase polypeptide under conditions which are conducive forproducing the engineered phenylalanine ammonia lyase polypeptide. Insome embodiments, the engineered phenylalanine ammonia lyase polypeptideis subsequently recovered from the resulting culture medium and/orcells.

The present invention provides exemplary engineered phenylalanineammonia lyase polypeptides having phenylalanine ammonia lyase activity.The Examples provide Tables showing sequence structural informationcorrelating specific amino acid sequence features with the functionalactivity of the engineered phenylalanine ammonia lyase polypeptides.This structure-function correlation information is provided in the formof specific amino acid residue differences relative to the referenceengineered polypeptide of SEQ ID NO: 2, 4, 8, 106, 252, 446, 482, 516,618, 714, 830, 894, 988, and/or 1140, as well as associatedexperimentally determined activity data for the exemplary engineeredphenylalanine ammonia lyase polypeptides.

In some embodiments, the engineered phenylalanine ammonia lyasepolypeptides of the present invention having phenylalanine ammonia lyaseactivity comprise an amino acid sequence having at least 85% sequenceidentity to reference sequence SEQ ID NO: 2, 4, 8, 106, 252, 446, 482,516, 618, 714, 830, 894, 988, and/or 1140, and which exhibits at leastone improved property, as compared to the reference sequence (e.g.,wild-type A. variabilis phenylalanine ammonia lyase).

In some embodiments the engineered phenylalanine ammonia lyasepolypeptides exhibiting at least one improved property have at least85%, at least 88%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or greater amino acid sequence identity with SEQ IDNO: 2, 4, 8, 106, 252, 446, 482, 516, 618, 714, 830, 894, 988, and/or1140, and an amino acid residue difference at one or more amino acidpositions (such as at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20or more amino acid positions) compared to SEQ ID NO: 2, 4, 8, 106, 252,446, 482, 516, 618, 714, 830, 894, 988, and/or 1140. In someembodiments, the engineered phenylalanine ammonia lyase polypeptide is apolypeptide listed in the Tables provided in the Examples.

In some embodiments, the present invention provides functional fragmentsof engineered phenylalanine ammonia lyase polypeptides. In someembodiments, functional fragments comprise at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% of the activity of the engineered phenylalanineammonia lyase polypeptide from which it was derived (i.e., the parentengineered phenylalanine ammonia lyase). In some embodiments, functionalfragments comprise at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% of the parent sequence of the engineered phenylalanine ammonialyase. In some embodiments the functional fragment will be truncated byless than 5, less than 10, less than 15, less than 10, less than 25,less than 30, less than 35, less than 40, less than 45, and less than 50amino acids.

In some embodiments, the present invention provides functional fragmentsof engineered phenylalanine ammonia lyase polypeptides. In someembodiments, functional fragments comprise at least about 95%, 96%, 97%,98%, or 99% of the activity of the engineered phenylalanine ammonialyase polypeptide from which it was derived (i.e., the parent engineeredphenylalanine ammonia lyase). In some embodiments, functional fragmentscomprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ofthe parent sequence of the engineered phenylalanine ammonia lyase. Insome embodiments the functional fragment will be truncated by less than5, less than 10, less than 15, less than 10, less than 25, less than 30,less than 35, less than 40, less than 45, less than 50, less than 55,less than 60, less than 65, or less than 70 amino acids.

In some embodiments, the engineered phenylalanine ammonia lyasepolypeptides exhibiting at least one improved property have at least85%, at least 88%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or greater amino acid sequence identity with SEQ IDNO: 2, 4, 8, 106, 252, 446, 482, 516, 618, 714, 830, 894, 988, and/or1140, and an amino acid residue difference at one or more amino acidpositions (such as at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 ormore amino acid positions) compared to SEQ ID NO: 2, 4, 8, 106, 252,446, 482, 516, 618, 714, 830, 894, 988, and/or 1140. In someembodiments, the engineered phenylalanine ammonia lyases comprise atleast 90% sequence identity to SEQ ID NO: 2, 4, 8, 106, 252, 446, 482,516, 618, 714, 830, 894, 988, and/or 1140, and comprise an amino aciddifference of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidpositions. In some embodiments, the engineered phenylalanine ammonialyase polypeptide consists of the sequence of SEQ ID NO: 4, 8, 106, 252,446, 482, 516, 618, 714, 830, 894, 988, and/or 1140.

Engineered PAL Polynucleotides Encoding Engineered Polypeptides,Expression Vectors and Host Cells

The present invention provides polynucleotides encoding the engineeredenzyme polypeptides described herein. In some embodiments, thepolynucleotides are operatively linked to one or more heterologousregulatory sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide. Insome embodiments, expression constructs containing at least oneheterologous polynucleotide encoding the engineered enzymepolypeptide(s) is introduced into appropriate host cells to express thecorresponding enzyme polypeptide(s).

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode an engineered enzyme (e.g., PAL) polypeptide. Thus,the present invention provides methods and compositions for theproduction of each and every possible variation of enzymepolynucleotides that could be made that encode the enzyme polypeptidesdescribed herein by selecting combinations based on the possible codonchoices, and all such variations are to be considered specificallydisclosed for any polypeptide described herein, including the amino acidsequences presented in the Examples (e.g., in the various Tables).

In some embodiments, the codons are preferably optimized for utilizationby the chosen host cell for protein production. For example, preferredcodons used in bacteria are typically used for expression in bacteria.Consequently, codon optimized polynucleotides encoding the engineeredenzyme polypeptides contain preferred codons at about 40%, 50%, 60%,70%, 80%, 90%, or greater than 90% of the codon positions in the fulllength coding region.

In some embodiments, the enzyme polynucleotide encodes an engineeredpolypeptide having enzyme activity with the properties disclosed herein,wherein the polypeptide comprises an amino acid sequence having at least60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequenceselected from SEQ ID NO: 2, 4, 8, 106, 252, 446, 482, 516, 618, 714,830, 894, 988, and/or 1140, or the amino acid sequence of any variant(e.g., those provided in the Examples), and one or more residuedifferences as compared to the reference polynucleotide(s), or the aminoacid sequence of any variant as disclosed in the Examples (for example1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions). Insome embodiments, the reference polypeptide sequence is selected fromSEQ ID NO: 2, 4, 8, 106, 482, 516, 618, 714, 830, 894, 988, and/or 1140.

In some embodiments, the phenylalanine ammonia lyase polynucleotideencodes an engineered polypeptide having phenylalanine ammonia lyaseactivity with the properties disclosed herein, wherein the polypeptidecomprises an amino acid sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentity to a reference sequence selected from SEQ ID NO: 4, 8, 106,252, 446, 482, 516, 618, 714, 830, 894, 988, and/or 1140, or the aminoacid sequence of any variant (e.g., those provided in the Examples), andone or more differences as compared to the reference polynucleotide ofSEQ ID NO: 1, 3, 7, 105, 251, 445, 481, 515, 617, 713, 829, 893, 987,and/or 1140, or the amino acid sequence of any variant as disclosed inthe Examples (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more aminoacid residue positions). In some embodiments, the reference sequence isselected from SEQ ID NO: 1, 3, 7, 105, 251, 445, 481, 515, 617, 713,829, 893, 987, and/or 1140. In some embodiments, the engineeredphenylalanine ammonia lyase variants comprise a polypeptide sequence setforth in SEQ ID NO: 4, 8, 106, 252, 446, 482, 516, 618, 714, 830, 894,988 and/or 1140. In some embodiments, the engineered phenylalanineammonia lyase variants comprise the substitution(s) or substitutionset(s) provided in the Examples (e.g., Tables 4.1, 5.1, 6.1, 7.1, 8.1,9.1, 10.1, 11.1, 12.1, 13.1, 14.1, 15.1, 18.1, and/or 19.1).

The present invention provides polynucleotides encoding the engineeredphenylalanine ammonia lyase variants provided herein. In someembodiments, the polynucleotides comprise a nucleotide sequence havingat least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identity to a reference sequence selectedfrom SEQ ID NO: 1, 3, 7, 105, 251, 445, 481, 515, 617, 713, 829, 893,987, and/or 1139, or the amino acid sequence of any variant (e.g., thoseprovided in the Examples), and one or more residue differences ascompared to the reference polynucleotide of SEQ ID NO: 1, 3, 7, 105,251, 445, 481, 515, 617, 713, 829, 893, 987, and/or 1139, or the aminoacid sequence of any variant as disclosed in the Examples (for example1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions). Insome embodiments, the reference sequence is selected from SEQ ID NO: 1,3, 7, 105, 251, 445, 481, 515, 617, 713, 829, 893, 987, and/or 1139. Insome embodiments, the polynucleotides are capable of hybridizing underhighly stringent conditions to a reference polynucleotide sequenceselected from SEQ ID NO: 1, 3, 7, 105, 251, 445, 481, 515, 617, 713,829, 893, 987, and/or 1139, or a complement thereof, or a polynucleotidesequence encoding any of the variant phenylalanine ammonia lyasepolypeptides provided herein. In some embodiments, the polynucleotidecapable of hybridizing under highly stringent conditions encodes aphenylalanine ammonia lyase polypeptide comprising an amino acidsequence that has one or more residue differences as compared to SEQ IDNO: 1, 3, 7, 105, 251, 445, 481, 515, 617, 713, 829, 893, 987, and/or1139. In some embodiments, the engineered phenylalanine ammonia lyasevariants are encoded by a polynucleotide sequence set forth in SEQ IDNO: 1, 3, 7, 105, 251, 445, 481, 515, 617, 713, 829, 893, 987, and/or1139.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference polynucleotide sequenceselected from any polynucleotide sequence provided herein, or acomplement thereof, or a polynucleotide sequence encoding any of thevariant enzyme polypeptides provided herein. In some embodiments, thepolynucleotide capable of hybridizing under highly stringent conditionsencodes an enzyme polypeptide comprising an amino acid sequence that hasone or more residue differences as compared to a reference sequence.

In some embodiments, an isolated polynucleotide encoding any of theengineered enzyme polypeptides herein is manipulated in a variety ofways to facilitate expression of the enzyme polypeptide. In someembodiments, the polynucleotides encoding the enzyme polypeptidescomprise expression vectors where one or more control sequences ispresent to regulate the expression of the enzyme polynucleotides and/orpolypeptides. Manipulation of the isolated polynucleotide prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector utilized. Techniques for modifying polynucleotides andnucleic acid sequences utilizing recombinant DNA methods are well knownin the art. In some embodiments, the control sequences include amongothers, promoters, leader sequences, polyadenylation sequences,propeptide sequences, signal peptide sequences, and transcriptionterminators. In some embodiments, suitable promoters are selected basedon the host cells selection. For bacterial host cells, suitablepromoters for directing transcription of the nucleic acid constructs ofthe present disclosure, include, but are not limited to promotersobtained from the E. coli lac operon, Streptomyces coelicolor agarasegene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacilluslicheniformis alpha-amylase gene (amyL), Bacillus stearothermophilusmaltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylasegene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillussubtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Seee.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731[1978]), as well as the tac promoter (See e.g., DeBoer et al., Proc.Natl Acad. Sci. USA 80: 21-25 [1983]). Exemplary promoters forfilamentous fungal host cells, include, but are not limited to promotersobtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusariumoxysporum trypsin-like protease (See e.g., WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof. Exemplary yeast cell promoters can be from the genes can befrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.Other useful promoters for yeast host cells are known in the art (Seee.g., Romanos et al., Yeast 8:423-488 [1992]).

In some embodiments, the control sequence is also a suitabletranscription terminator sequence (i.e., a sequence recognized by a hostcell to terminate transcription). In some embodiments, the terminatorsequence is operably linked to the 3′ terminus of the nucleic acidsequence encoding the enzyme polypeptide. Any suitable terminator whichis functional in the host cell of choice finds use in the presentinvention. Exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease. Exemplary terminators for yeast hostcells can be obtained from the genes for Saccharomyces cerevisiaeenolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomycescerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other usefulterminators for yeast host cells are known in the art (See e.g., Romanoset al., supra).

In some embodiments, the control sequence is also a suitable leadersequence (i.e., a non-translated region of an mRNA that is important fortranslation by the host cell). In some embodiments, the leader sequenceis operably linked to the 5′ terminus of the nucleic acid sequenceencoding the enzyme polypeptide. Any suitable leader sequence that isfunctional in the host cell of choice find use in the present invention.Exemplary leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulanstriose phosphate isomerase. Suitable leaders for yeast host cells areobtained from the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

In some embodiments, the control sequence is also a polyadenylationsequence (i.e., a sequence operably linked to the 3′ terminus of thenucleic acid sequence and which, when transcribed, is recognized by thehost cell as a signal to add polyadenosine residues to transcribedmRNA). Any suitable polyadenylation sequence which is functional in thehost cell of choice finds use in the present invention. Exemplarypolyadenylation sequences for filamentous fungal host cells include, butare not limited to the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase. Useful polyadenylation sequences for yeast hostcells are known (See e.g., Guo and Sherman, Mol. Cell. Bio.,15:5983-5990 [1995]).

In some embodiments, the control sequence comprises a signal peptide(i.e., a coding region that codes for an amino acid sequence linked tothe amino terminus of a polypeptide and directs the encoded polypeptideinto the cell's secretory pathway). In some embodiments, the 5′ end ofthe coding sequence of the nucleic acid sequence inherently contains asignal peptide coding region naturally linked in translation readingframe with the segment of the coding region that encodes the secretedpolypeptide. Alternatively, in some embodiments, the 5′ end of thecoding sequence contains a signal peptide coding region that is foreignto the coding sequence. Any suitable signal peptide coding region whichdirects the expressed polypeptide into the secretory pathway of a hostcell of choice finds use for expression of the engineeredpolypeptide(s). Effective signal peptide coding regions for bacterialhost cells are the signal peptide coding regions include, but are notlimited to those obtained from the genes for Bacillus NClB 11837maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacilluslicheniformis subtilisin, Bacillus licheniformis beta-lactamase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are known in the art(See e.g., Simonen and Palva, Microbiol. Rev., 57:109-137 [1993]). Insome embodiments, effective signal peptide coding regions forfilamentous fungal host cells include, but are not limited to the signalpeptide coding regions obtained from the genes for Aspergillus oryzaeTAKA amylase, Aspergillus niger neutral amylase, Aspergillus nigerglucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolenscellulase, and Humicola lanuginosa lipase. Useful signal peptides foryeast host cells include, but are not limited to those from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase.

In some embodiments, the control sequence is also a propeptide codingregion that codes for an amino acid sequence positioned at the aminoterminus of a polypeptide. The resultant polypeptide is referred to as a“proenzyme,” “propolypeptide,” or “zymogen.” A propolypeptide can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from any suitable source, including, butnot limited to the genes for Bacillus subtilis alkaline protease (aprE),Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiaealpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthorathermophila lactase (See e.g., WO 95/33836). Where both signal peptideand propeptide regions are present at the amino terminus of apolypeptide, the propeptide region is positioned next to the aminoterminus of a polypeptide and the signal peptide region is positionednext to the amino terminus of the propeptide region.

In some embodiments, regulatory sequences are also utilized. Thesesequences facilitate the regulation of the expression of the polypeptiderelative to the growth of the host cell. Examples of regulatory systemsare those that cause the expression of the gene to be turned on or offin response to a chemical or physical stimulus, including the presenceof a regulatory compound. In prokaryotic host cells, suitable regulatorysequences include, but are not limited to the lac, tac, and trp operatorsystems. In yeast host cells, suitable regulatory systems include, butare not limited to the ADH2 system or GAL1 system. In filamentous fungi,suitable regulatory sequences include, but are not limited to the TAKAalpha-amylase promoter, Aspergillus niger glucoamylase promoter, andAspergillus oryzae glucoamylase promoter.

In another aspect, the present invention is directed to a recombinantexpression vector comprising a polynucleotide encoding an engineeredenzyme polypeptide, and one or more expression regulating regions suchas a promoter and a terminator, a replication origin, etc., depending onthe type of hosts into which they are to be introduced. In someembodiments, the various nucleic acid and control sequences describedherein are joined together to produce recombinant expression vectorswhich include one or more convenient restriction sites to allow forinsertion or substitution of the nucleic acid sequence encoding theenzyme polypeptide at such sites. Alternatively, in some embodiments,the nucleic acid sequence of the present invention is expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Insome embodiments involving the creation of the expression vector, thecoding sequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any suitable vector (e.g., aplasmid or virus), that can be conveniently subjected to recombinant DNAprocedures and bring about the expression of the enzyme polynucleotidesequence. The choice of the vector typically depends on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

In some embodiments, the expression vector is an autonomouslyreplicating vector (i.e., a vector that exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, such as a plasmid, an extra-chromosomal element, aminichromosome, or an artificial chromosome). The vector may contain anymeans for assuring self-replication. In some alternative embodiments,the vector is one in which, when introduced into the host cell, it isintegrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, in someembodiments, a single vector or plasmid, or two or more vectors orplasmids which together contain the total DNA to be introduced into thegenome of the host cell, and/or a transposon is utilized.

In some embodiments, the expression vector contains one or moreselectable markers, which permit easy selection of transformed cells. A“selectable marker” is a gene, the product of which provides for biocideor viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markersinclude, but are not limited to the dal genes from Bacillus subtilis orBacillus licheniformis, or markers, which confer antibiotic resistancesuch as ampicillin, kanamycin, chloramphenicol or tetracyclineresistance. Suitable markers for yeast host cells include, but are notlimited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectablemarkers for use in filamentous fungal host cells include, but are notlimited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae),argB (ornithine carbamoyltransferases), bar (phosphinothricinacetyltransferase; e.g., from S. hygroscopicus), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase; e.g., from A. nidulans or A.orzyae), sC (sulfate adenyltransferase), and trpC (anthranilatesynthase), as well as equivalents thereof.

In another aspect, the present invention provides a host cell comprisingat least one polynucleotide encoding at least one engineered enzymepolypeptide of the present invention, the polynucleotide(s) beingoperatively linked to one or more control sequences for expression ofthe engineered enzyme enzyme(s) in the host cell. Host cells suitablefor use in expressing the polypeptides encoded by the expression vectorsof the present invention are well known in the art and include but arenot limited to, bacterial cells, such as E. coli, Vibrio fluvialis,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowesmelanoma cells; and plant cells. Exemplary host cells also includevarious Escherichia coli strains (e.g., W3110 (ΔfhuA) and BL21).Examples of bacterial selectable markers include, but are not limited tothe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol, and or tetracycline resistance.

In some embodiments, the expression vectors of the present inventioncontain an element(s) that permits integration of the vector into thehost cell's genome or autonomous replication of the vector in the cellindependent of the genome. In some embodiments involving integrationinto the host cell genome, the vectors rely on the nucleic acid sequenceencoding the polypeptide or any other element of the vector forintegration of the vector into the genome by homologous or nonhomologousrecombination.

In some alternative embodiments, the expression vectors containadditional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements preferably contain a sufficient number ofnucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000base pairs, and most preferably 800 to 10,000 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori or the origins of replication of plasmids pBR322, pUC19, pACYC177(which plasmid has the P15A ori), or pACYC184 permitting replication inE. coli, and pUB110, pE194, or pTA1060 permitting replication inBacillus. Examples of origins of replication for use in a yeast hostcell are the 2 micron origin of replication, ARS1, ARS4, the combinationof ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin ofreplication may be one having a mutation which makes it's functioningtemperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl.Acad. Sci. USA 75:1433 [1978]).

In some embodiments, more than one copy of a nucleic acid sequence ofthe present invention is inserted into the host cell to increaseproduction of the gene product. An increase in the copy number of thenucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the nucleic acidsequence where cells containing amplified copies of the selectablemarker gene, and thereby additional copies of the nucleic acid sequence,can be selected for by cultivating the cells in the presence of theappropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors include,but are not limited to the p3×FLAG™™ expression vectors (Sigma-AldrichChemicals), which include a CMV promoter and hGH polyadenylation sitefor expression in mammalian host cells and a pBR322 origin ofreplication and ampicillin resistance markers for amplification in E.coli. Other suitable expression vectors include, but are not limited topBluescriptII SK(−) and pBK-CMV (Stratagene), and plasmids derived frompBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly(See e.g., Lathe et al., Gene 57:193-201 [1987]).

Thus, in some embodiments, a vector comprising a sequence encoding atleast one variant phenylalanine ammonia lyase is transformed into a hostcell in order to allow propagation of the vector and expression of thevariant phenylalanine ammonia lyase(s). In some embodiments, the variantphenylalanine ammonia lyases are post-translationally modified to removethe signal peptide and in some cases may be cleaved after secretion. Insome embodiments, the transformed host cell described above is culturedin a suitable nutrient medium under conditions permitting the expressionof the variant phenylalanine ammonia lyase(s). Any suitable mediumuseful for culturing the host cells finds use in the present invention,including, but not limited to minimal or complex media containingappropriate supplements. In some embodiments, host cells are grown inHTP media. Suitable media are available from various commercialsuppliers or may be prepared according to published recipes (e.g., incatalogues of the American Type Culture Collection).

In another aspect, the present invention provides host cells comprisinga polynucleotide encoding an improved phenylalanine ammonia lyasepolypeptide provided herein, the polynucleotide being operatively linkedto one or more control sequences for expression of the phenylalanineammonia lyase enzyme in the host cell. Host cells for use in expressingthe phenylalanine ammonia lyase polypeptides encoded by the expressionvectors of the present invention are well known in the art and includebut are not limited to, bacterial cells, such as E. coli, Bacillusmegaterium, Lactobacillus kefir, Streptomyces and Salmonella typhimuriumcells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiaeor Pichia pastoris (ATCC Accession No. 201178)); insect cells such asDrosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS,BHK, 293, and Bowes melanoma cells; and plant cells. Appropriate culturemedia and growth conditions for the above-described host cells are wellknown in the art.

Polynucleotides for expression of the phenylalanine ammonia lyase may beintroduced into cells by various methods known in the art. Techniquesinclude among others, electroporation, biolistic particle bombardment,liposome mediated transfection, calcium chloride transfection, andprotoplast fusion. Various methods for introducing polynucleotides intocells are known to those skilled in the art.

In some embodiments, the host cell is a eukaryotic cell. Suitableeukaryotic host cells include, but are not limited to, fungal cells,algal cells, insect cells, and plant cells. Suitable fungal host cellsinclude, but are not limited to, Ascomycota, Basidiomycota,Deuteromycota, Zygomycota, Fungi imperfecti. In some embodiments, thefungal host cells are yeast cells and filamentous fungal cells. Thefilamentous fungal host cells of the present invention include allfilamentous forms of the subdivision Eumycotina and Oomycota.Filamentous fungi are characterized by a vegetative mycelium with a cellwall composed of chitin, cellulose and other complex polysaccharides.The filamentous fungal host cells of the present invention aremorphologically distinct from yeast.

In some embodiments of the present invention, the filamentous fungalhost cells are of any suitable genus and species, including, but notlimited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus,Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis,Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora,Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces,Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium,Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes,Tolypocladium, Trichoderma, Verticillium, and/or Volvariella, and/orteleomorphs, or anamorphs, and synonyms, basionyms, or taxonomicequivalents thereof.

In some embodiments of the present invention, the host cell is a yeastcell, including but not limited to cells of Candida, Hansenula,Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowiaspecies. In some embodiments of the present invention, the yeast cell isHansenula polymorpha, Saccharomyces cerevisiae, Saccharomycescarlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis,Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia quercuum, Pichia ptjperi, Pichia stipitis, Pichiamethanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, orYarrowia lipolytica.

In some embodiments of the invention, the host cell is an algal cellsuch as Chlamydomonas (e.g., C. reinhardtii) and Phormidium (P. sp.ATCC29409).

In some other embodiments, the host cell is a prokaryotic cell. Suitableprokaryotic cells include, but are not limited to Gram-positive,Gram-negative and Gram-variable bacterial cells. Any suitable bacterialorganism finds use in the present invention, including but not limitedto Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter,Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium,Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter,Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia,Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium,Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter,Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus,Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium,Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus,Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia,Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus,Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella,Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula,Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella,Yersinia and Zymomonas. In some embodiments, the host cell is a speciesof Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium,Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium,Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus,Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella,Streptococcus, Streptomyces, or Zymomonas. In some embodiments, thebacterial host strain is non-pathogenic to humans. In some embodimentsthe bacterial host strain is an industrial strain. Numerous bacterialindustrial strains are known and suitable in the present invention. Insome embodiments of the present invention, the bacterial host cell is anAgrobacterium species (e.g., A. radiobacter, A. rhizogenes, and A.rubi). In some embodiments of the present invention, the bacterial hostcell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A.globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A.paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfureus, and A.ureafaciens). In some embodiments of the present invention, thebacterial host cell is a Bacillus species (e.g., B. thuringensis, B.anthracia, B. megaterium, B. subtilis, B. lentus, B. circulars, B.pumilus, B. lautus, B.coagulans, B. brevis, B. firmus, B. alkaophius, B.licheniformis, B. clausii, B. stearothermophilus, B. halodurans, and B.amyloliquefaciens). In some embodiments, the host cell is an industrialBacillus strain including but not limited to B. subtilis, B. pumilus, B.licheniformis, B. megaterium, B. clausii, B. stearothermophilus, or B.amyloliquefaciens. In some embodiments, the Bacillus host cells are B.subtilis, B. licheniformis, B. megaterium, B. stearothermophilus, and/orB. amyloliquefaciens. In some embodiments, the bacterial host cell is aClostridium species (e.g., C. acetobutylicum, C. tetani E88, C.lituseburense, C. saccharobutylicum, C. perfringens, and C.betjerinckii). In some embodiments, the bacterial host cell is aCorynebacterium species (e.g., C. glutamicum and C. acetoacidophilum).In some embodiments the bacterial host cell is an Escherichia species(e.g., E. coli). In some embodiments, the host cell is Escherichia coliW3110. In some embodiments, the bacterial host cell is an Erwiniaspecies (e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E.punctata, and E. terreus). In some embodiments, the bacterial host cellis a Pantoea species (e.g., P. citrea, and P. agglomerans). In someembodiments the bacterial host cell is a Pseudomonas species (e.g., P.putida, P. aeruginosa, P. mevalonii, and P. sp. D-0l 10). In someembodiments, the bacterial host cell is a Streptococcus species (e.g.,S. equisimiles, S. pyogenes, and S. uberis). In some embodiments, thebacterial host cell is a Streptomyces species (e.g., S. ambofaciens, S.achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus,S. fungicidicus, S. griseus, and S. lividans). In some embodiments, thebacterial host cell is a Zymomonas species (e.g., Z. mobilis, and Zlipolytica).

Many prokaryotic and eukaryotic strains that find use in the presentinvention are readily available to the public from a number of culturecollections such as American Type Culture Collection (ATCC), DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (DSM), CentraalbureauVoor Schimmelcultures (CBS), and Agricultural Research Service PatentCulture Collection, Northern Regional Research Center (NRRL).

In some embodiments, host cells are genetically modified to havecharacteristics that improve protein secretion, protein stability and/orother properties desirable for expression and/or secretion of a protein.Genetic modification can be achieved by genetic engineering techniquesand/or classical microbiological techniques (e.g., chemical or UVmutagenesis and subsequent selection). Indeed, in some embodiments,combinations of recombinant modification and classical selectiontechniques are used to produce the host cells. Using recombinanttechnology, nucleic acid molecules can be introduced, deleted, inhibitedor modified, in a manner that results in increased yields ofphenylalanine ammonia lyase variant(s) within the host cell and/or inthe culture medium. For example, knockout of Alp1 function results in acell that is protease deficient, and knockout of pyr5 function resultsin a cell with a pyrimidine deficient phenotype. In one geneticengineering approach, homologous recombination is used to inducetargeted gene modifications by specifically targeting a gene in vivo tosuppress expression of the encoded protein. In alternative approaches,siRNA, antisense and/or ribozyme technology find use in inhibiting geneexpression. A variety of methods are known in the art for reducingexpression of protein in cells, including, but not limited to deletionof all or part of the gene encoding the protein and site-specificmutagenesis to disrupt expression or activity of the gene product. (Seee.g., Chaveroche et al., Nucl. Acids Res., 28:22 e97 [2000]; Cho et al.,Molec. Plant Microbe Interact., 19:7-15 [2006]; Maruyama and Kitamoto,Biotechnol Lett., 30:1811-1817 [2008]; Takahashi et al., Mol. Gen.Genom., 272: 344-352 [2004]; and You et al., Arch. Micriobiol.,191:615-622 [2009], all of which are incorporated by reference herein).Random mutagenesis, followed by screening for desired mutations alsofinds use (See e.g., Combier et al., FEMS Microbiol. Lett., 220:141-8[2003]; and Firon et al., Eukary. Cell 2:247-55 [2003], both of whichare incorporated by reference).

Introduction of a vector or DNA construct into a host cell can beaccomplished using any suitable method known in the art, including butnot limited to calcium phosphate transfection, DEAE-dextran mediatedtransfection, PEG-mediated transformation, electroporation, or othercommon techniques known in the art. In some embodiments, the Escherichiacoli expression vector pCK100900i (See, U.S. Pat. No. 9,714,437, whichis hereby incorporated by reference herein) finds use.

In some embodiments, the engineered host cells (i.e., “recombinant hostcells”) of the present invention are cultured in conventional nutrientmedia modified as appropriate for activating promoters, selectingtransformants, or amplifying the phenylalanine ammonia lyasepolynucleotide. Culture conditions, such as temperature, pH and thelike, are those previously used with the host cell selected forexpression, and are well-known to those skilled in the art. As noted,many standard references and texts are available for the culture andproduction of many cells, including cells of bacterial, plant, animal(especially mammalian) and archaebacterial origin.

In some embodiments, cells expressing the variant phenylalanine ammonialyase polypeptides of the invention are grown under batch or continuousfermentations conditions. Classical “batch fermentation” is a closedsystem, wherein the compositions of the medium is set at the beginningof the fermentation and is not subject to artificial alternations duringthe fermentation. A variation of the batch system is a “fed-batchfermentation” which also finds use in the present invention. In thisvariation, the substrate is added in increments as the fermentationprogresses. Fed-batch systems are useful when catabolite repression islikely to inhibit the metabolism of the cells and where it is desirableto have limited amounts of substrate in the medium. Batch and fed-batchfermentations are common and well known in the art. “Continuousfermentation” is an open system where a defined fermentation medium isadded continuously to a bioreactor and an equal amount of conditionedmedium is removed simultaneously for processing. Continuous fermentationgenerally maintains the cultures at a constant high density where cellsare primarily in log phase growth. Continuous fermentation systemsstrive to maintain steady state growth conditions. Methods formodulating nutrients and growth factors for continuous fermentationprocesses as well as techniques for maximizing the rate of productformation are well known in the art of industrial microbiology.

In some embodiments of the present invention, cell-freetranscription/translation systems find use in producing variantphenylalanine ammonia lyase(s). Several systems are commerciallyavailable and the methods are well-known to those skilled in the art.

The present invention provides methods of making variant phenylalanineammonia lyase polypeptides or biologically active fragments thereof. Insome embodiments, the method comprises: providing a host celltransformed with a polynucleotide encoding an amino acid sequence thatcomprises at least about 70% (or at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%) sequence identity to SEQ ID NO: 2, 4, 8, 106, 482, 516, 618, 714,830, 894, 988, and/or 1140, and comprising at least one mutation asprovided herein; culturing the transformed host cell in a culture mediumunder conditions in which the host cell expresses the encoded variantphenylalanine ammonia lyase polypeptide; and optionally recovering orisolating the expressed variant phenylalanine ammonia lyase polypeptide,and/or recovering or isolating the culture medium containing theexpressed variant phenylalanine ammonia lyase polypeptide. In someembodiments, the methods further provide optionally lysing thetransformed host cells after expressing the encoded phenylalanineammonia lyase polypeptide and optionally recovering and/or isolating theexpressed variant phenylalanine ammonia lyase polypeptide from the celllysate. The present invention further provides methods of making avariant phenylalanine ammonia lyase polypeptide comprising cultivating ahost cell transformed with a variant phenylalanine ammonia lyasepolypeptide under conditions suitable for the production of the variantphenylalanine ammonia lyase polypeptide and recovering the variantphenylalanine ammonia lyase polypeptide. Typically, recovery orisolation of the phenylalanine ammonia lyase polypeptide is from thehost cell culture medium, the host cell or both, using protein recoverytechniques that are well known in the art, including those describedherein. In some embodiments, host cells are harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. Microbial cells employed inexpression of proteins can be disrupted by any convenient method,including, but not limited to freeze-thaw cycling, sonication,mechanical disruption, and/or use of cell lysing agents, as well as manyother suitable methods well known to those skilled in the art.

Engineered phenylalanine ammonia lyase enzymes expressed in a host cellcan be recovered from the cells and/or the culture medium using any oneor more of the techniques known in the art for protein purification,including, among others, lysozyme treatment, sonication, filtration,salting-out, ultra-centrifugation, and chromatography. Suitablesolutions for lysing and the high efficiency extraction of proteins frombacteria, such as E. coli, are commercially available under the tradename CelLytic B™ (Sigma-Aldrich). Thus, in some embodiments, theresulting polypeptide is recovered/isolated and optionally purified byany of a number of methods known in the art. For example, in someembodiments, the polypeptide is isolated from the nutrient medium byconventional procedures including, but not limited to, centrifugation,filtration, extraction, spray-drying, evaporation, chromatography (e.g.,ion exchange, affinity, hydrophobic interaction, chromatofocusing, andsize exclusion), or precipitation. In some embodiments, proteinrefolding steps are used, as desired, in completing the configuration ofthe mature protein. In addition, in some embodiments, high performanceliquid chromatography (HPLC) is employed in the final purificationsteps. For example, in some embodiments, methods known in the art, finduse in the present invention (See e.g., Parry et al., Biochem. J.,353:117 [2001]; and Hong et al., Appl. Microbiol. Biotechnol., 73:1331[2007], both of which are incorporated herein by reference). Indeed, anysuitable purification methods known in the art find use in the presentinvention.

Chromatographic techniques for isolation of the phenylalanine ammonialyase polypeptide include, but are not limited to reverse phasechromatography high performance liquid chromatography, ion exchangechromatography, gel electrophoresis, and affinity chromatography.Conditions for purifying a particular enzyme will depend, in part, onfactors such as net charge, hydrophobicity, hydrophilicity, molecularweight, molecular shape, etc., are known to those skilled in the art.

In some embodiments, affinity techniques find use in isolating theimproved phenylalanine ammonia lyase enzymes. For affinitychromatography purification, any antibody which specifically binds thephenylalanine ammonia lyase polypeptide may be used. For the productionof antibodies, various host animals, including but not limited torabbits, mice, rats, etc., may be immunized by injection with thephenylalanine ammonia lyase. The phenylalanine ammonia lyase polypeptidemay be attached to a suitable carrier, such as BSA, by means of a sidechain functional group or linkers attached to a side chain functionalgroup. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(Bacillus Calmette Guerin) and Corynebacterium parvum.

In some embodiments, the phenylalanine ammonia lyase variants areprepared and used in the form of cells expressing the enzymes, as crudeextracts, or as isolated or purified preparations. In some embodiments,the phenylalanine ammonia lyase variants are prepared as lyophilisates,in powder form (e.g., acetone powders), or prepared as enzyme solutions.In some embodiments, the phenylalanine ammonia lyase variants are in theform of substantially pure preparations.

In some embodiments, the phenylalanine ammonia lyase polypeptides areattached to any suitable solid substrate. Solid substrates include butare not limited to a solid phase, surface, and/or membrane. Solidsupports include, but are not limited to organic polymers such aspolystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled pore glass (CPG), reverse phase silica or metal, such as goldor platinum. The configuration of the substrate can be in the form ofbeads, spheres, particles, granules, a gel, a membrane or a surface.Surfaces can be planar, substantially planar, or non-planar. Solidsupports can be porous or non-porous, and can have swelling ornon-swelling characteristics. A solid support can be configured in theform of a well, depression, or other container, vessel, feature, orlocation. A plurality of supports can be configured on an array atvarious locations, addressable for robotic delivery of reagents, or bydetection methods and/or instruments.

In some embodiments, immunological methods are used to purifyphenylalanine ammonia lyase variants. In one approach, antibody raisedagainst a variant phenylalanine ammonia lyase polypeptide (e.g., againsta polypeptide comprising any of SEQ ID NO: 2, 4, 8, 106, 482, 516, 618,714, 830, 894, 988, and 1140, and/or an immunogenic fragment thereof)using conventional methods is immobilized on beads, mixed with cellculture media under conditions in which the variant phenylalanineammonia lyase is bound, and precipitated. In a related approach,immunochromatography finds use.

In some embodiments, the variant phenylalanine ammonia lyases areexpressed as a fusion protein including a non-enzyme portion. In someembodiments, the variant phenylalanine ammonia lyase sequence is fusedto a purification facilitating domain. As used herein, the term“purification facilitating domain” refers to a domain that mediatespurification of the polypeptide to which it is fused. Suitablepurification domains include, but are not limited to metal chelatingpeptides, histidine-tryptophan modules that allow purification onimmobilized metals, a sequence which binds glutathione (e.g., GST), ahemagglutinin (HA) tag (corresponding to an epitope derived from theinfluenza hemagglutinin protein; See e.g., Wilson et al., Cell 37:767[1984]), maltose binding protein sequences, the FLAG epitope utilized inthe FLAGS extension/affinity purification system (e.g., the systemavailable from Immunex Corp), and the like. One expression vectorcontemplated for use in the compositions and methods described hereinprovides for expression of a fusion protein comprising a polypeptide ofthe invention fused to a polyhistidine region separated by anenterokinase cleavage site. The histidine residues facilitatepurification on IMIAC (immobilized metal ion affinity chromatography;See e.g., Porath et al., Prot. Exp. Purif., 3:263-281 [1992]) while theenterokinase cleavage site provides a means for separating the variantphenylalanine ammonia lyase polypeptide from the fusion protein. pGEXvectors (Promega) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to ligand-agarose beads (e.g., glutathione-agarose in thecase of GST-fusions) followed by elution in the presence of free ligand.

Accordingly, in another aspect, the present invention provides methodsof producing the engineered enzyme polypeptides, where the methodscomprise culturing a host cell capable of expressing a polynucleotideencoding the engineered enzyme polypeptide under conditions suitable forexpression of the polypeptide. In some embodiments, the methods furthercomprise the steps of isolating and/or purifying the enzymepolypeptides, as described herein.

Appropriate culture media and growth conditions for host cells are wellknown in the art. It is contemplated that any suitable method forintroducing polynucleotides for expression of the enzyme polypeptidesinto cells will find use in the present invention. Suitable techniquesinclude, but are not limited to electroporation, biolistic particlebombardment, liposome mediated transfection, calcium chloridetransfection, and protoplast fusion.

Various features and embodiments of the present invention areillustrated in the following representative examples, which are intendedto be illustrative, and not limiting.

EXPERIMENTAL

The following Examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present invention. Indeed, there are various suitablesources for many of the reagents and equipment described below. It isnot intended that the present invention be limited to any particularsource for any reagent or equipment item.

In the experimental disclosure below, the following abbreviations apply:M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol(moles); gm and g (gram); mg (milligrams); ug and μg (micrograms); L and1 (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters);um and μm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) andhr(s) (hour(s)); U (units); MW (molecular weight); AUC (area undercurve); rpm (rotations per minute); psi and PSI (pounds per squareinch); ° C. (degrees Centigrade); RT and rt (room temperature); CV(coefficient of variability); CAM and cam (chloramphenicol); PMBS(polymyxin B sulfate); IPTG (isopropyl β-D-1-thiogalactopyranoside); LB(lysogeny broth); TB (terrific broth); SFP (shake flask powder); CDS(coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid);nt (nucleotide; polynucleotide); aa (amino acid; polypeptide); E. coliW3110 (commonly used laboratory E. coli strain, available from the ColiGenetic Stock Center [CGSC], New Haven, Conn.); HTP (high throughput);HPLC (high pressure liquid chromatography); HPLC-UV (HPLC-UltravioletVisible Detector); 1H NMR (proton nuclear magnetic resonancespectroscopy); FIOPC (fold improvements over positive control); Sigmaand Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.; Difco (DifcoLaboratories, BD Diagnostic Systems, Detroit, Mich.); Microfluidics(Microfluidics, Westwood, Mass.); Life Technologies (Life Technologies,a part of Fisher Scientific, Waltham, Mass.); Amresco (Amresco, LLC,Solon, Ohio); Carbosynth (Carbosynth, Ltd., Berkshire, UK); Varian(Varian Medical Systems, Palo Alto, Calif.); Agilent (AgilentTechnologies, Inc., Santa Clara, Calif.); Infors (Infors USA Inc.,Annapolis Junction, Md.); and Thermotron (Thermotron, Inc., Holland,Mich.).

Example 1 Preparation of HTP PAL Containing Wet Cell Pellets

A synthetic gene (SEQ ID NO: 1) encoding Anabaena variabilisphenylalanine ammonia lyase (AvPAL) (SEQ ID NO: 2) optimized forexpression in E. coli was cloned into a pCK110900 vector. An evolvedvariant of wild-type AvPAL (SEQ ID NO: 2) that was more stable and hadtyrosine ammonia lyase activity was chosen as the parent gene (SEQ IDNO: 4). W3110 E. coli cells were transformed with the respective plasmidcontaining the parent PAL encoding gene (SEQ ID NO: 3) and plated on LBagar plates containing 1% glucose and 30 μg/ml chloramphenicol (CAM),and grown overnight at 37° C. Monoclonal colonies were picked andinoculated into 180 μl LB containing 1% glucose and 30 μg/mLchloramphenicol and placed in the wells of 96-well shallow-wellmicrotiter plates. The plates were sealed with O₂-permeable seals andcultures were grown overnight at 30° C., 200 rpm and 85% humidity. Then,10 μl of each of the cell cultures were transferred into the wells of96-well deep-well plates containing 390 μl TB and 30 μg/mL CAM. Thedeep-well plates were sealed with O₂-permeable seals and incubated at30° C., 250 rpm and 85% humidity until OD₆₀₀ 0.6-0.8 was reached. Thecell cultures were then induced by adding isopropyl thioglycoside (IPTG)to a final concentration of 1 mM and incubated overnight at 30° C. with250 rpm shaking. The cells were then pelleted using centrifugation at4,000 rpm for 10 min. The supernatants were discarded and the pelletsfrozen at −80° C. prior to lysis.

Example 2 Preparation of HTP PAL-Containing Cell Lysates

Frozen pellets prepared as described in EXAMPLE 1 were lysed with 400 μllysis buffer containing 100 mM triethanolamine buffer, pH 7.5, 1 g/Llysozyme and 0.5 g/L. The lysis mixture was shaken at room temperaturefor 2 hours. The plate was then centrifuged for 15 min at 4000 rpm and4° C. The supernatants were then used in biocatalytic reactions asclarified lysate to determine enzymatic activity.

Example 3 Preparation of Lyophilized Lysates from Shake Flask (SF)Cultures

A single colony containing the desired gene picked from an LB agarplates with 1% glucose and 30 μg/ml CAM, and incubated overnight at 37°C. was transferred to 6 ml of LB with 1% glucose and 30 μg/ml CAM. Theculture was grown for 18 h at 30° C., 250 rpm, and subculturedapproximately 1:50 into 250 ml of TB containing 30 μg/ml CAM, to a finalOD₆₀₀ of about 0.05. The subculture was grown for approximately 195minutes at 30° C., 250 rpm, to an OD₆₀₀ between 0.6-0.8, and inducedwith 1 mM IPTG. The subculture was then grown for 20 h at 30° C. and 250rpm. The subculture was centrifuged at 4000 rpm for 20 min. Thesupernatant was discarded, and the pellet was resuspended in 35 ml of 25mM triethanolamine buffer, pH 7.5. The cells were lysed using aMicrofluidizer® processor system (Microfluidics) at 18,000 psi. Thelysate was pelleted (10,000 rpm×60 min), and the supernatant was frozenand lyophilized to generate shake flake (SF) enzyme powder.

Example 4 Improved PAL Variants for Production of Compound 2

A variant of wild type PAL from Anabaena variabilis (SEQ ID NO: 2) waschosen as the initial parent enzyme (SEQ ID NO: 4). Libraries ofengineered genes were produced using well-established techniques (e.g.,saturation mutagenesis, and recombination of previously identifiedbeneficial mutations). The polypeptides encoded by each gene wereproduced in HTP as described in EXAMPLE 1, and the clarified lysateswere generated as described in EXAMPLE 2.

Each 100 μL reaction was carried out in 96-well shallow well microtiterplates with 50% (v/v) clarified cell lysate, 10 mM compound (1), 1 Mammonium carbonate, pH˜9. The plates were heat sealed and incubated at30° C. and agitated at 500 RPM in an Infors Thermotron® shakerovernight. The plate was removed and quenched by adding 1 volume (100μL) of methanol to each well followed by mixing and centrifugation. Thesupernatant was then diluted an additional amount in methanol as neededto be above the limit of detection and within the linear range of theanalysis. The analysis was performed on the Agilent RapidFire 365 highthroughput mass spectrometer using the manufacturer's protocols.

Activity relative to SEQ ID NO: 4 was calculated as the area under thecurve of the product formed by the variant, as compared to that of SEQID NO: 4, as determined by the previously described RapidFire analysis.

TABLE 4.1 Relative Activities of PAL Variants on Production of Compound2 from Compound 1 (Relative to SEQ ID NO: 4) SEQ ID NO: Amino AcidDifferences FIOP (activity) (nt/aa) (Relative to SEQ ID NO: 4) (Relativeto SEQ ID NO: 4)¹ 5/6 V80A/L104A/V105I/M222V +++ 7/8L104A/A220G/M222V/H359Y +++ 9/10 L104A/H359Y +++ 11/12 V80A/L104A +++13/14 L104A/S175A/A220G/M222V ++ 15/16 V80A/E99D/L104A/S175A/A220G/H359Y++ 17/18 V80A/L104A/V105I/A220G ++ 19/20E99D/L104A/V105I/V172T/S175A/A220G/M222V ++ 21/22V80A/L104A/V105I/A220G/M222V/M416V ++ 23/24 V80A/L104A/H359Y/M416V ++25/26 V80A/L104A/V172A/S175A/A220G/I310A/H359Y + 27/28L104A/V105I/S175A + 29/30 L104A/V172A/I310A/H359Y + 31/32V80A/L104A/V172T/M222V + 33/34 V80A/L104A/V105I/V172A/A220G/M222V +35/36 L104A/S175A/L213Q/M222V/H359Y + 37/38V80A/L104A/V105I/V172T/S175A/M222V/H359Y + 39/40V80A/L104A/V172A/S175A + 41/42 V80A/L104A/V105I/V172A + 43/44 L104I +++45/46 L104A +++ 47/48 L100R +++ 49/50 L100S +++ 51/52 H107T +++ 53/54N451P +++ 55/56 L219G ++ 57/58 F84V ++ 59/60 L219P ++ 61/62 M416L ++63/64 L219M/E540G ++ 65/66 L104S ++ 67/68 G360V ++ 69/70 F84P ++ 71/72I423E + 73/74 Q452A + 75/76 M416E + 77/78 L418G + 79/80 L108E + 81/82S175G/S315R + 83/84 T110P/K419D + 85/86 L104P + 87/88 N347V + 89/90V90T + 91/92 Q101K + 93/94 A220P + 95/96 S405R + 97/98 F363R +  99/100F450E + ¹Levels of increased activity were determined relative to thereference polypeptide of SEQ ID NO: 4, and defined as follows: ““+”” =2.05 to 3.93 (first 50%); ““++”” >3.93 (next 30%); and ““+++”” >27.51(top 20%).

Example 5 Improved PAL Variants for Production of Compound 2

SEQ ID NO: 8 was selected as the parent enzyme for the next round ofevolution. Libraries of engineered genes were produced usingwell-established techniques (e.g., saturation mutagenesis, andrecombination of previously identified beneficial mutations). Thepolypeptides encoded by each gene were produced in HTP as described inEXAMPLE 1, and the clarified lysates were generated as described inEXAMPLE 2. HTP screening reactions were carried out as described inEXAMPLE 4. Activity relative to SEQ ID NO: 8 was calculated as describedin EXAMPLE 4.

TABLE 5.1 Relative Activities of PAL Variants on Production of Compound2 from Compound 1 (Relative to SEQ ID NO: 8) SEQ ID FIOP (activity) NO:Amino Acid Differences (Relative to SEQ (nt/aa) (Relative to SEQ ID NO:8) ID NO: 8)¹ 101/102 A97T/V105I/H107G/G111A/V222G/L421T/C424V +++103/104 T102E/V105I/H107S/V222G/Y304W/A394N/L421T/C424V +++ 105/106T102E/V105I/H107S/V222G/Y304W/G307H/A394N/L421T/C424V +++ 107/108T102E/H107A/G111A/V222G/A394N +++ 109/110G83S/T102E/V105I/W106R/H107G/M416V/G420S/L421T +++ 111/112V105I/H107G/G111A/A394N/G420S/C424V +++ 113/114 M416G +++ 115/116T102E/V105I/H107G/A394N/M416V/C424V +++ 117/118V80A/T102E/V105I/H107L/Y304W +++ 119/120 F84P +++ 121/122G83S/T102E/V105I/A394N/M416V/G420S +++ 123/124G74D/T102E/V105I/W106R/H107L/S175A/A394N +++ 125/126G74D/A97T/V105I/W106R/H107G +++ 127/128 V105I/H107A/V222G/Y304W/M416V+++ 129/130 G74D/V80A/V105I/H107G/A394N/G420S ++ 131/132G74D/T102E/V105I/W106R/H107G/S175A/A394N/L421T ++ 133/134 L219C ++135/136 F84G ++ 137/138 V105I/W106R/H107G/G420S/L421T ++ 139/140A97T/T102E/V105I/W106R/H107G/G111A/A394N/G420S/L421T ++ 141/142T102E/H107G/G420S/C424V ++ 143/144 H107G/L421T ++ 145/146H107L/V222G/Y304W ++ 147/148G74D/G83S/T102E/V105I/H107S/G111A/V222A/A394N/M416V ++ 149/150V105I/H107I/G111A/Y304W ++ 151/152 H107T/G111A/S2091/V222G/Y304W ++153/154 A97T/T102E/G111A/S175A/V222G/G420S/L421T ++ 155/156A97T/V105I/H107S/G111A/A394N/M416V/L421T ++ 157/158Y304W/A394N/M416V/G420S ++ 159/160 V105I/H107E/G111A ++ 161/162A97T/T102E/V105I/H107G/G111A/S175A/Y304W/L421T/C424V ++ 163/164F84V/E99D/A1041/V105I/L219G ++ 165/166 T102E/V105I/H1071/Y304W/C424V ++167/168 H107G ++ 169/170 V222A/L421T/C424V ++ 171/172 S175N ++ 173/174A104G ++ 175/176 V80A/F84V/E99D/A1041/V105I/H107T/L219G + 177/178H107L + 179/180 N103S + 181/182 K216G + 183/184 F84V/E99D + 185/186H107Q + 187/188 F84V/H1071 + 189/190 H107G/S291N + 191/192 F84V +193/194 F84L + 195/196 M416H + 197/198 M416V + 199/200 G420A + 201/202M416A + 203/204 K413E + 205/206 D306L + 207/208 A394V + 209/210V105I/H107T + 211/212 V105I + 213/214 F84S + 215/216 F84R + 217/218G20D/D306L/P564Q + 219/220 H107P + 221/222 W106M + 223/224 S395M +225/226 E99D/V105I/H107T + 227/228 V105I/L219G + 229/230 K413T + 231/232V105I/S175A/L219G + 233/234 Y359R + 235/236 M416C + 237/238 T102N +239/240 G420S + 241/242 V105I/G111A/L219G + 243/244 M416L + 245/246L418I + 247/248 G220S + 249/250 H107T + ¹Levels of increased activitywere determined relative to the reference polypeptide of SEQ ID NO: 8and defined as follows: ““+”” = 2.02 to 7.09 (first 50%); ““++”” >7.09(next 30%); and ““+++”” >23.66 (top 20%).

Example 6 Improved PAL Variants for Production of Compound 2

SEQ ID NO: 106 was selected as the parent enzyme for the next round ofevolution. Libraries of engineered genes were produced usingwell-established techniques (e.g., saturation mutagenesis, andrecombination of previously identified beneficial mutations). Thepolypeptides encoded by each gene were produced in HTP as described inEXAMPLE 1, and the clarified lysates were generated as described inEXAMPLE 2. HTP screening reactions were carried out as described inEXAMPLE 4, except that the lysate was diluted 2-fold before adding tothe reaction plate and the reaction temperature was increased to 40° C.Activity relative to SEQ ID NO: 106 was calculated as described inEXAMPLE 4.

TABLE 6.1 Relative Activities of PAL Variants on Production of Compound2 from Compound 1 (Relative to SEQ ID NO: 106) SEQ ID FIOP (activity)NO: Amino Acid Differences (Relative to (nt/aa) (Relative to SEQ ID NO:106) SEQ ID NO: 106)¹ 251/252 L219C/G220S +++ 253/254 G220S/R410K +++255/256 G220S/Y359R +++ 257/258 S107G/G220S +++ 259/260 S107A +++261/262 L4S +++ 263/264 P76T +++ 265/266 P76H +++ 267/268 R410K +++269/270 S107G/K216G/R410K +++ 271/272 S107A/L219C +++ 273/274L219C/G220S/R410K +++ 275/276 T3E +++ 277/278 P76L ++ 279/280 P76M ++281/282 K10S ++ 283/284 D303I ++ 285/286 Q6D ++ 287/288 F84P/S107A/L219C++ 289/290 P76R ++ 291/292 T3P ++ 293/294 R40D ++ 295/296 L566G ++297/298 T3E/A550T ++ 299/300 S5D ++ 301/302 A7D ++ 303/304 S107G ++305/306 S22V/P76S ++ 307/308 T3K ++ 309/310 T3R ++ 311/312 L4P ++313/314 E102N/S107G/L219C/R410K ++ 315/316 A24V + 317/318 A502Q +319/320 S5P + 321/322 A7G + 323/324 F84P/S107G/L219C + 325/326 T3H +327/328 H567D + 329/330 P76E + 331/332 Q14A + 333/334 D303T + 335/336N25R + 337/338 T212P + 339/340 K10A + 341/342 K301S + 343/344 D303V +345/346 A7T + 347/348 Q6S + 349/350 D303K + 351/352 S286R + 353/354P76A + 355/356 A502T + 357/358 P76L/D561L + 359/360 S5L + 361/362 K10P +363/364 D303R + 365/366 R544W + 367/368 E75L + 369/370 T212N + 371/372T3N + 373/374 S22A + 375/376 S107G/L219C + 377/378F84P/S107A/S175A/L219C + ¹Levels of increased activity were determinedrelative to the reference polypeptide of SEQ ID NO: 106, and defined asfollows: ““+”” = 1.49 to 2.77 (first 50%); ““++”” >2.77 (next 30%); and““+++”” >4.88 (top 20%).

Example 7 Improved PAL Variants for Production of Compound 2

SEQ ID NO: 252 was selected as the parent enzyme for the next round ofevolution. Libraries of engineered genes were produced usingwell-established techniques (e.g., saturation mutagenesis, andrecombination of previously identified beneficial mutations). Thepolypeptides encoded by each gene were produced in HTP as described inEXAMPLE 1, and the clarified lysates were generated as described inEXAMPLE 2. HTP screening reactions were carried out as described inEXAMPLE 6, except that the lysate was diluted 8-fold before adding tothe reaction plate. Activity relative to SEQ ID NO: 252 was calculatedas described in EXAMPLE 4.

TABLE 7.1 Relative Activities of PAL Variants on Production of Compound2 from Compound 1 (Relative to SEQ ID NO: 252) SEQ ID NO: Amino AcidDifferences FIOP (activity) (nt/aa) (Relative to SEQ ID NO: 252)(Relative to SEQ ID NO: 252)¹ 379/380 P76M/F84P/S107G/H307G +++ 381/382S107A/H307G +++ 383/384 F84P/H307G +++ 385/386 P76M/F84P/S107A/H307G +++387/388 P76M/F84P/S107A +++ 389/390 P76T/F84P/S107G +++ 391/392P76L/S107A/H307G +++ 393/394 P76M/F84P/S107G ++ 395/396F84P/S107A/H307G/A502Q ++ 397/398 P76L/S107G/H307G ++ 399/400 H307G ++401/402 P76M/S107G ++ 403/404 F84P/K301S/H307G/L566G ++ 405/406F84P/S107A/H307G ++ 407/408 P76T/S107G ++ 409/410 H307G/A502Q ++ 411/412P76M/S107A ++ 413/414 P76T + 415/416T3E/L4S/S5P/A7G/P76T/F84P/S107A/H307G + 417/418 P76L/H307G + 419/420A24V/P76A/S107A/H307G + 421/422 S107A/A502Q/L566G + 423/424A24V/F84P/S107G/H307G + 425/426 H307G/L566G + 427/428P76A/F84P/S107A/A502Q + 429/430 S107A/K301S/A502Q + 431/432P76H/F84P/S107A + 433/434 S107A/A502Q + 435/436 T3E/A7G/F84P/H307G +437/438 P76H/H307G/A502Q + 439/440 T3K/A7D/P76L/S107A/H307G/L566G +441/442 S107A/H307G/L566G + 443/444 P76T/F84P/S107A/H307G/A502Q +445/446 T3P/S5P/S107A/H307G/L566G + ¹Levels of increased activity weredetermined relative to the reference polypeptide of SEQ ID NO: 252 anddefined as follows: ““+”” = 3.51 to 6.29 (first 50%); ““++”” >6.29 (next30%); and ““+++”” >7.44 (top 20%).

Example 8 Improved PAL Variants for Production of Compound 2

SEQ ID NO: 446 was selected as the parent enzyme for the next round ofevolution. Libraries of engineered genes were produced usingwell-established techniques (e.g., saturation mutagenesis, andrecombination of previously identified beneficial mutations). Thepolypeptides encoded by each gene were produced in HTP as described inEXAMPLE 1, and the clarified lysates were generated as described inEXAMPLE 2. HTP screening reactions were carried out as described inEXAMPLE 7. Activity relative to SEQ ID NO: 446 was calculated asdescribed in EXAMPLE 4.

TABLE 8.1 Relative Activities of PAL Variants on Production of Compound2 from Compound 1 (Relative to SEQ ID NO: 446) SEQ ID FIOP (activity)NO: Amino Acid Differences (Relative to (nt/aa) (Relative to SEQ ID NO:446) SEQ ID NO: 446)¹ 447/448 P3E/Q6S/A502Q +++ 449/450 D303I/A502Q ++451/452 P3H/L4S/A7G ++ 453/454 P76A ++ 455/456 P76R/A502Q ++ 457/458D303I ++ 459/460 P3H/A7D/P76R ++ 461/462 P3E/L4S/A7V/P76H ++ 463/464P3K/L4S/Q6S/A7V/D303I ++ 465/466 P3H/A7D/P76H + 467/468 R40D/D303I +469/470 P76R + 471/472 P3T/L4S/Q6D/A7D/P76R + 473/474 A7G/D303I +475/476 P76H + 477/478 P76L + 479/480 G222V +++ 481/482 E102M +++483/484 G222T +++ 485/486 S82T +++ 487/488 L171P +++ 489/490 G222T/E509K++ 491/492 L100H ++ 493/494 L171V + 495/496 L174G + 497/498 C219T +499/500 C219M + 501/502 T345S + 503/504 K216G + 505/506 G218A + 507/508W304H + 509/510 W304V + 511/512 W304F + ¹Levels of increased activitywere determined relative to the reference polypeptide of SEQ ID NO: 446,and defined as follows: ““+”” = 1.47 to 2.19 (first 50%); ““++””

Example 9 Improved PAL Variants for Production of Compound 2

SEQ ID NO: 482 was selected as the parent enzyme for the next round ofevolution. Libraries of engineered genes were produced usingwell-established techniques (e.g., saturation mutagenesis, andrecombination of previously identified beneficial mutations). Thepolypeptides encoded by each gene were produced in HTP as described inEXAMPLE 1, and the clarified lysates were generated as described inEXAMPLE 2. HTP screening reactions were carried out as described inEXAMPLE 7, except that the lysate was diluted 4-fold before adding tothe reaction, the concentration of compound (1) was increased to 40 mM,and 2 M ammonium carbamate was used in place of the 1 M ammoniumcarbonate. Activity relative to SEQ ID NO: 482 was calculated asdescribed in EXAMPLE 4.

TABLE 9.1 Relative Activities of PAL Variants on Production of Compound2 from Compound 1 (Relative to SEQ ID NO: 482) SEQ ID FIOP (activity)NO: Amino Acid Differences (Relative to (nt/aa) (Relative to SEQ ID NO:482) SEQ ID NO: 482)¹ 513/514 A7D/P76L/S82T/L174G/G222T/D303I +++515/516 A7D/P76M/L174G/G222V +++ 517/518 L174G/G222V +++ 519/520A7D/S82T +++ 521/522 P76L/S82T/K216G/G218A +++ 523/524 S82T +++ 525/526P76M/K216G +++ 527/528 P76M/K216G/C219T +++ 529/530 R40D/S82T +++531/532 P3E/L4S/A7D ++ 533/534 L171P ++ 535/536 L4S/A7D/K216G/G218A ++537/538 A112L ++ 539/540 T460F ++ 541/542 A7D/K216G/G218A ++ 543/544 A7D++ 545/546 P3E/A7D ++ 547/548 C219T ++ 549/550 F443Q ++ 551/552A7D/P76M/C219T/D303I ++ 553/554 L4S/A7D ++ 555/556 K216G ++ 557/558A7D/R40D ++ 559/560 G222V + 561/562 I428L + 563/564 C219T/T345S +565/566 L538V + 567/568 N437H + 569/570 A543Q + 571/572 S271A + 573/574L47P + 575/576 Q366S + 577/578 S524D + 579/580 A112S + 581/582 N474Y +583/584 S524A + 585/586 S524R + 587/588 A112T + 589/590 I268T + 591/592L47Q + 593/594 I428M + 595/596 C503T + 597/598 Y66W + 599/600 L538I +601/602 S3311 + 603/604 S209A + ¹Levels of increased activity weredetermined relative to the reference polypeptide of SEQ ID NO: 482, anddefined as follows: ““+”” = 1.38 to 2.41 (first 50%); ““++”” >2.41 (next30%); and ““+++”” >3.94 (top 20%).

Example 10 Improved PAL Variants for Production of Compound 2

SEQ ID NO: 516 was selected as the parent enzyme for the next round ofevolution. Libraries of engineered genes were produced usingwell-established techniques (e.g., saturation mutagenesis, andrecombination of previously identified beneficial mutations). Thepolypeptides encoded by each gene were produced in HTP as described inEXAMPLE 1, and the clarified lysates were generated as described inEXAMPLE 2. HTP screening reactions were carried out as described inEXAMPLE 9, except that the lysate concentration was changed to 5% v:vand was not diluted before adding to the reaction and the ammoniumcarbamate concentration was increased to 4 M. Activity relative to SEQID NO: 516 was calculated as described in EXAMPLE 4.

TABLE 10.1 Relative Activities of PAL Variants on Production of Compound2 from Compound 1 (Relative to SEQ ID NO: 516) SEQ ID FIOP (activity)NO: Amino Acid Differences (Relative to (nt/aa) (Relative to SEQ ID NO:516) SEQ ID NO: 516)¹ 605/606 R94P/V554R +++ 607/608 R94P/I149T +++609/610 M76H +++ 611/612 L47K/R94P/E509L +++ 613/614 S524A +++ 615/616S271A/T345S +++ 617/618 F84P +++ 619/620 W304S +++ 621/622 S271A/I428L+++ 623/624 N44H/R94P/N270Q/V554R +++ 625/626 M76H/T345S ++ 627/628L47P/I428L ++ 629/630 L47P/M76H/T345S ++ 631/632 M76H/S271A ++ 633/634L47P ++ 635/636 D303I ++ 637/638 N44H/R94P/V554R ++ 639/640 L4I/W304C ++641/642 N44H/L47K ++ 643/644 L47K ++ 645/646 N44H/L47K/R94P/E509L ++647/648 A112L/S524A ++ 649/650 R40D ++ 651/652 L47P/M76H ++ 653/654R40D/N437H ++ 655/656 R94P/K195E ++ 657/658 D7S + 659/660L47K/K195E/V554R + 661/662 W304H + 663/664 W304L + 665/666 N25A +667/668 D306K + 669/670 T51A/W106G + 671/672 K413T + 673/674S98N/T460A + 675/676 F16Y + 677/678 S98E + 679/680 Q6R + 681/682 R410M +683/684 S82T + 685/686 K109G + 687/688 L4G + 689/690 S9C + 691/692M416T + 693/694 N25G + 695/696 Y358L + 697/698 S98A + 699/700 L3491 +701/702 G20T + 703/704 H302R + 705/706 F16S + 707/708 K413S + ¹Levels ofincreased activity were determined relative to the reference polypeptideof SEQ ID NO: 516, and defined as follows: ““+”” = 1.74 to 3.80 (first50%); ““++”” >3.80 (next 30%); and ““+++”” >7.04 (top 20%).

Example 11 Improved PAL Variants for Production of Compound 2

SEQ ID NO: 618 was selected as the parent enzyme for the next round ofevolution. Libraries of engineered genes were produced usingwell-established techniques (e.g., saturation mutagenesis, andrecombination of previously identified beneficial mutations). Thepolypeptides encoded by each gene were produced in HTP as described inEXAMPLE 1, and the clarified lysates were generated as described inEXAMPLE 2. HTP screening reactions were carried out as described inEXAMPLE 10, except the lysate concentration was changed to 10% v:v andwas diluted 4-fold before adding to the reaction and the ammoniumcarbamate concentration was increased to 4.5 M. Activity relative to SEQID NO: 618 was calculated as described in EXAMPLE 4.

TABLE 11.1 Relative Activities of PAL Variants on Production of Compound2 from Compound 1 (Relative to SEQ ID NO: 618) SEQ ID FIOP (activity)NO: Amino Acid Differences (Relative to (nt/aa) (Relative to SEQ ID NO:618) SEQ ID NO: 618)¹ 709/710 L47K/M76H/R94P/S271A +++ 711/712F16Y/N44H/R94P/S98E/S524A +++ 713/714 L47P/M76H/W304S/S524A/V554R +++715/716 L47K/M76H/W304S/D306K/V554R +++ 717/718 R40D/M76H/W304S/N437H+++ 719/720 M76H/W304S/N437H +++ 721/722 L47K/R94P/S271A/W304S/V554R +++723/724 R94P/S98E/S524A +++ 725/726 R40D/N44H/M76H/W304H/E509L +++727/728 D7S/R94P/S98E +++ 729/730 L47K/M76H/S82T/S271A/W304S +++ 731/732L47K/M76H/S82T/R94P/S271A ++ 733/734 L4I/L47K/M76H/S82T/R94P ++ 735/736L47K/R94P ++ 737/738 L47K/R94P/S271A ++ 739/740 M76H/S271A/W304S/V554R++ 741/742 R94P/S98N ++ 743/744 D7S/N44H/R94P/S98N ++ 745/746R94P/S98E/E509L ++ 747/748 N44H/M76H/A112L ++ 749/750L47P/M76H/R94P/S271A/D306K/L375M/S524A/V554R ++ 751/752F16Y/N44H/M76H/S98E ++ 753/754 R94P/S98E/D306K ++ 755/756D7S/L47P/M76H/S82T ++ 757/758 S98E/N270Q/W304S/V554R ++ 759/760N44H/M76H/R94P/A112L/W304S ++ 761/762 R94P/V554R ++ 763/764R40D/N44H/S98A/W304S ++ 765/766 F16Y/R40D/M76H + 767/768N44H/R94P/S271A/W304S/N437H/V554R + 769/770 R40D/M76H/V554R + 771/772D7S/M76H/V554R + 773/774 T54K/N68A/Y158H/S209E/T212Q/T495A/H517E +775/776 M76H + 777/778 N25T/T54K/N68A/Y158H/I339V/H517E + 779/780N25T/T54K/N68A/E72A + 781/782 N25T/T54E/Y158H/S209E/T212Q/I339V/A551S +783/784 N30G/N68A/E72A/N207G/5209E/T212Q/I339V/T495A/H517E + 785/786N25T/T54E/N68A/E72A/5209E/T212Q/I339V/H517E + 787/788 S82T/V554R +789/790 N25T/T54E/N68A/E72A/Y158H/I339V + 791/792N68A/E72A/Y158H/H517E + 793/794 N68A/Y158H/5209E/T495A/H517E/A5515 +795/796 N25T/T54E/E72A/I339V/H517E + 797/798N68A/E72A/Y158H/5209E/T212Q/I339V/T495A/A5515 + 799/800 582T + 801/802N400A + 803/804 S49R/N114K/Q240K/Q521K + 805/806 A119E + 807/808 V294A +809/810 S3571 + 811/812 R516M + 813/814 A119V + 815/816 R527V + 817/818C565E + 819/820 V294C + ¹Levels of increased activity were determinedrelative to the reference polypeptide of SEQ ID NO: 618, and defined asfollows: ““+”” = 1.61 to 4.89 (first 50%); ““++””

Example 12 Improved PAL Variants for Production of Compound 2

SEQ ID NO: 714 was selected as the parent enzyme for the next round ofevolution. Libraries of engineered genes were produced usingwell-established techniques (e.g., saturation mutagenesis, andrecombination of previously identified beneficial mutations). Thepolypeptides encoded by each gene were produced in HTP as described inEXAMPLE 1, and the clarified lysates were generated as described inEXAMPLE 2. HTP screening reactions were carried out as described inEXAMPLE 11, except that the lysate was not diluted before adding to thereaction, the concentration of compound (1) was increased to 80 mM, andthe ammonium carbamate concentration was increased to 5 M. Activityrelative to SEQ ID NO: 714 was calculated as described in EXAMPLE 4.

TABLE 12.1 Relative Activities of PAL Variants on Production of Compound2 from Compound 1 (Relative to SEQ ID NO: 714) SEQ ID Amino AcidDifferences FIOP (activity) NO: (nt/aa) (Relative to SEQ ID NO: 714)(Relative to SEQ ID NO: 714)¹ 821/822 N207G; R410M; T460A; H517E +++823/824 Y158H; S209E; R410M; H517E +++ 825/826 N68A; I339V; H517E +++827/828 N25T; T54E; S271A; H517E +++ 829/830 T460A +++ 831/832 E72A;Y158H; S209E; R410M; H517E +++ 833/834 N25T; D306E; I339V +++ 835/836R40D; P47K; N68A; R94P; S98A; R410M; H517E ++ 837/838 N25T; Y158H;S209E; R410M ++ 839/840 E72A; R94P; Y158H; I339V; R410M; T460A; H517E ++841/842 R94P; Y158H; S209E; I339V; R410M ++ 843/844 R410M; H517E ++845/846 N25T; R40D; Y158H; S209E; S304H; R410M; H517E ++ 847/848 T54E;N68A; E72A; S98E; S209E; H517E ++ 849/850 G83L ++ 851/852 R40D; N68A;T460A; H517E ++ 853/854 I339V ++ 855/856 G83P ++ 857/858 R317E + 859/860R410M + 861/862 N25T; R410M + 863/864 S220A + 865/866 N207G; R410M +867/868 P47K; S98E; I339V; R410M + 869/870 Y158H; N207G; I339V; R410M +871/872 T460A; H517E + 873/874 N25T + 875/876 Y158H + 877/878 M416I +879/880 N394S + 881/882 L100G + 883/884 S220A + 885/886 H517E + 887/888A1291 + 889/890 N68A + 891/892 G83P + ¹Levels of increased activity weredetermined relative to the reference polypeptide of SEQ ID NO: 714, anddefined as follows: ““+”” = 1.27 to 1.67 (first 50%); ““++”” >1.67 (next30%); and ““+++”” >1.90 (top 20%).

Example 13 Improved PAL Variants for Production of Compound 2

SEQ ID NO: 830 was selected as the parent enzyme. Libraries ofengineered genes were produced using well-established techniques (e.g.,saturation mutagenesis, and recombination of previously identifiedbeneficial mutations). The polypeptides encoded by each gene wereproduced in HTP as described in EXAMPLE 1, and the clarified lysateswere generated as described in EXAMPLE 2. HTP screening reactions werecarried out as described in EXAMPLE 12, except that the ammoniumcarbamate concentration was changed to 4.5 M. Activity relative to SEQID NO: 830 was calculated as described in EXAMPLE 4.

TABLE 13.1 Relative Activities of PAL Variants on Production of Compound2 from Compound 1 (Relative to SEQ ID NO: 830) SEQ ID FIOP (activity)NO: Amino Acid Differences (Relative to (nt/aa) (Relative to SEQ ID NO:830) SEQ ID NO: 830)¹ 893/894 G83P/S209E/S220A/R410M/H517E +++ 895/896S220A/H517E +++ 897/898 N25T/Y158H/S220A +++ 899/900N25T/Y158H/S220A/H517E +++ 901/902 N25T/G83L/Y158H/S220A/H517E +++903/904 N25T/Y158H/S209E/S220A/H517E +++ 905/906 S271A/R410M/M4161/H517E+++ 907/908 S220A +++ 909/910 S220A/R410M/M4161/H517E +++ 911/912G83P/I339V/R410M ++ 913/914 N25T/R410M/M4161/H517E ++ 915/916N25T/G83P/S220A/M416I ++ 917/918 R410M/M4161/H517E ++ 919/920Y158H/S220A/S271A/H517E ++ 921/922 N25T/S220A/H517E ++ 923/924N25T/S220A/I339V ++ 925/926 I339V ++ 927/928 G45H ++ 929/930 G520A ++931/932 S209P ++ 933/934 G83P ++ 935/936 A246V ++ 937/938 S209T +939/940 A119Q + 941/942 N400Q + 943/944 T54K/I285L + 945/946 R40G +947/948 V424A + 949/950 T54K/G59R + 951/952 S271A + 953/954 Y459F +955/956 A244S + 957/958 S525P + 959/960 G537P + 961/962 A479S + 963/964L293M + 965/966 R410E + 967/968 C565K + 969/970 S304A + 971/972 R410A +973/974 G537A + 975/976 N400A + 977/978 V368F + 979/980 I562V + ¹Levelsof increased activity were determined relative to the referencepolypeptide of SEQ ID NO: 830 and defined as follows: ““+”” = 1.20 to1.60 (first 50%); ““++”” >1.60 (next 30%); and ““+++”” >2.05 (top 20%).

Example 14 Improved PAL Variants for Production of Compound 2

SEQ ID NO: 894 was selected as the parent enzyme. Libraries ofengineered genes were produced using well-established techniques (e.g.,saturation mutagenesis, and recombination of previously identifiedbeneficial mutations). The polypeptides encoded by each gene wereproduced in HTP as described in EXAMPLE 1, and the clarified lysateswere generated as described in EXAMPLE 2. HTP screening reactions werecarried out as described in EXAMPLE 13, except that the lysate wasdiluted 8-fold before adding to the reaction and the reaction wasanalyzed by HPLC as described in EXAMPLE 16. Activity relative to SEQ IDNO: 894 was calculated as the percent conversion of the variant comparedto the percent conversion of SEQ ID NO: 894.

TABLE 14.1 Relative Activities of PAL Variants on Production of Compound2 from Compound 1 (Relative to SEQ ID NO: 894) SEQ ID Amino AcidDifferences FIOP (activity) NO: (nt/aa) (Relative to SEQ ID NO: 894)(Relative to SEQ ID NO: 894) 981/982 T54K/E209P/L214N/A2448/I339V/C503T+++ 983/984 E209P/C503T +++ 985/986 T54P +++ 987/988 T54P/V424A +++989/990 A246V/V424A ++ 991/992 R40Q/T54P/E209P/L214N/A2448/I339V/G520A++ 993/994 N25T/R40C/V424A ++ 995/996 T54P/V424A/G520A ++ 997/998N25T/R40G/G45H/E209P/V424A ++  999/1000 R40Q/P47R/T54P/L214N/C503T ++1001/1002 R40G/E209P/A246V/V424A ++ 1003/1004 T54P/E209P/L214N/A244S ++1005/1006 R40T/T54P/L214N/A244S/I339V/C503T + 1007/1008N25T/T54P/S73K/E209T/V424A/G520A + 1009/1010N25T/G45H/T54L/S73K/A246V/V424A + 1011/1012 A246V + 1013/1014 V424A +1015/1016 V424C + 1017/1018 K413S + 1019/1020 V227F + 1021/1022 V424S +1023/1024 V424G + 1025/1026 H274P/Q311S + 1027/1028 M410Q + 1029/1030E411A + ¹Levels of increased activity were determined relative to thereference polypeptide of SEQ ID NO: 894, and defined as follows: ““+”” =1.21 to 1.64 (first 50%); ““++”” >1.64 (next 30%); and ““+++”” >1.81(top 20%).

Example 15 Improved PAL Variants for Production of Compound 2

SEQ ID NO: 988 was selected as the parent enzyme. Libraries ofengineered genes were produced using well-established techniques (e.g.,saturation mutagenesis, and recombination of previously identifiedbeneficial mutations). The polypeptides encoded by each gene wereproduced in HTP as described in EXAMPLE 1, and the clarified lysateswere generated as described in EXAMPLE 2. HTP screening reactions werecarried out as described in EXAMPLE 14. Activity relative to SEQ ID NO:988 was calculated as described in EXAMPLE 14.

TABLE 15.1 Relative Activities of PAL Variants on Production of Compound2 from Compound 1 (Relative to SEQ ID NO: 988) SEQ ID Amino AcidDifferences FIOP (activity) NO: (Relative to SEQ (Relative to (nt/aa) IDNO: 988) SEQ ID NO: 988)¹ 1031/1032 T463S +++ 1033/1034 I454V +++1035/1036 R40C/A424C +++ 1037/1038 R40C/V90Q/ +++ T421S/A424C 1039/1040T463G +++ 1041/1042 I454L +++ 1043/1044 P54L/L214Q ++ 1045/1046 R40C ++1047/1048 R40C/L214Q ++ 1049/1050 A424C ++ 1051/1052 T463A ++ 1053/1054R40C/L214N/A424C ++ 1055/1056 T463N ++ 1057/1058 R40C/T421S/A424G ++1059/1060 W106S/V227F/ ++ A244S/R554C 1061/1062 W106S/V227F/A244S ++1063/1064 T463W + 1065/1066 T463L + 1067/1068 L214Q + 1069/1070R40C/P54L/L214Q/ + T421S/A424C 1071/1072 T421S/A424C + 1073/1074 T463V +1075/1076 V90Q/L214Q/A424G + 1077/1078 W106S/V227F/R554C + 1079/1080L464C + 1081/1082 L214Q/T421S + 1083/1084 A543Q + 1085/1086W106R/V227F + 1087/1088 L464Q + 1089/1090 I339M + 1091/1092 Y66F +1093/1094 N474E + ¹Levels of increased activity were determined relativeto the reference polypeptide of SEQ ID NO: 988 and defined as follows:““+”” = 1.20 to 1.30 (first 50%); ““++”” >1.30 (next 30%); and““+++”” >1.43 (top 20%).

Example 16 HPLC Analytical Method for Monitoring Reaction in Scheme 2

This Example provides the methods used to collect the data provided inExamples 14, 15, 18 and 19. The methods provided in this Example finduse in analyzing the variants produced using the present invention.However, it is not intended that the present invention be limited to themethods described herein, as other suitable methods are known to thoseskilled in the art.

TABLE 16.1 Analytical Method Instrument Agilent HPLC 1200 series ColumnPhenomenex Onyx Monolithic C18 100 × 3 mm Mobile Phase A: water + 0.1%trifluoroacetic acid B: acetonitrile + 0.1% trifluoroacetic acid MobilePhase 0 min: 10% B Gradient 0-0.5 min ramp to 100% B 0.5-0.51 min rampto 10% B 1 min: stop Flow Rate 3 mL/min Run Time ~ 1 min Substrate andProduct Compound (1): 0.65 min Elution order Compound (2): 0.52 minColumn Temperature 50° C. Injection Volume 5 μL Detection UV at 350 nmfor compound (1) UV at 280 nm for compound (2) Conversion [AUC Compound(2)]/[AUC Compound calculation (2) + 3.2*AUC Compound (1)]

Example 17 Enantioselectivity of PAL Variants

This Example provides the method used to determine theenantioselectivity of the reaction shown in Scheme 2. Only some of thevariants described in Examples 4-15 were evaluated forenantioselectivity and in each case the undesired (R)-amino acid was notobserved under these conditions. The methods provided in this Examplefind use in analyzing the variants produced using the present invention.However, it is not intended that the present invention be limited to themethods described herein, as other suitable methods are known to thoseskilled in the art.

TABLE 17.1 Analytical Method Instrument Shimadzu LC20 HPLC series ColumnAstec Chirobiotic T, 250 × 4.6 mm × 5 μm Mobile Phase Methanol + 0.1%trimethylamine + 0.2% acetic acid, isocratic Flow Rate 2 mL/min Run Time6 min Substrate and Product Compound (2) (S)-isomer: 3.7 min Elutionorder Compound (2) (R)-isomer: 4.6 min Column Temperature 40° C.Injection Volume 10 μL Detection UV at 280 nm for compound

Example 18 Improved PAL Variants for Production of Compound 2

SEQ ID NO: 988 was selected as the parent enzyme for another next roundof evolution. Libraries of engineered genes were produced usingwell-established techniques (e.g., saturation mutagenesis, andrecombination of previously identified beneficial mutations). Thepolypeptides encoded by each gene were produced in HTP as described inEXAMPLE 1, and the clarified lysates were generated as described inEXAMPLE 2. HTP screening reactions were carried out as described inEXAMPLE 14. Activity relative to SEQ ID NO: 988 was calculated asdescribed in EXAMPLE 14.

TABLE 18.1 Relative Activities of PAL Variants on Production of Compound4 from Compound 3 (Relative to SEQ ID NO: 988) SEQ Amino Acid FIOP(activity) ID NO: Differences Relative to SEQ (nt/aa) (Relative to SEQID NO: 988) ID NO: 988¹ 1095/1096 E517D +++ 1097/1098 A524I +++1099/1100 A104G +++ 1101/1102 I105G +++ 1103/1104 A424G +++ 1105/1106P47E ++ 1107/1108 P47T ++ 1109/1110 R554L ++ 1111/1112 N394L ++1113/1114 T421Q ++ 1115/1116 M410T ++ 1117/1118 M410V ++ 1119/1120M410Y + 1121/1122 P47A + 1123/1124 P47R + 1125/1126 M410L + 1127/1128A424L + 1129/1130 R554V + 1131/1132 M102S + 1133/1134 G154T +++1135/1136 N36Q ++ 1137/1138 N36C + 1139/1140 R40C/Y66F/M410K/N474E +++1141/1142 Y66F/L214Q/A424C +++ 1143/1144 A244S/E411A +++ 1145/1146M410K/E411A/A424G ++ 1147/1148 Y66F/V227F ++ 1149/1150Y66F/V227F/A244S/A424C/A543Q ++ 1151/1152 R40C/Y66F/V227F ++ 1153/1154Y66F/T463L/L464Q ++ 1155/1156 Y66F/L214Q/N437G/N474E ++ 1157/1158L214Q/A244S/A543Q ++ 1159/1160 Y66F/M370E + 1161/1162Y66F/M410K/A424C/I454V/R527H + 1163/1164 Y66F/I339L + 1165/1166Y66F/A424C + 1167/1168 L214Q/H374D/A424C + 1169/1170Y66F/L214Q/H374D/M410K/N474E + 1171/1172 Y66F/I339L/M410K/A543Q +1173/1174 V227F/A244S/E411A/A424C + 1175/1176 V227F/I339L/K413A/N437G +1177/1178 Y66F/V227F/A424G + 1179/1180 Y66F/A543Q + 1181/1182R40C/Y66F/V227F/A244S/M410K/A424C + 1183/1184 Y66F/I339L/N474E +1185/1186 R40C/M410K/E411A/A424C + 1187/1188 Y66F/T463A/L464C +1189/1190 I339L +++ 1191/1192 Y66F + 1193/1194 K4131 + 1195/1196 V227F +¹Levels of increased activity were determined relative to the referencepolypeptide of SEQ ID NO: 988, and defined as follows: “+” = 1.27 to1.44 (first 50%); “++” >1.44 (next 30%); and “+++” >1.55 (top 20%).

Example 19 Improved PAL Variants for Production of Compound 2

SEQ ID NO: 1140 was selected as the parent enzyme for another round ofevolution. Libraries of engineered genes were produced usingwell-established techniques (e.g., saturation mutagenesis, andrecombination of previously identified beneficial mutations). Thepolypeptides encoded by each gene were produced in HTP as described inEXAMPLE 1, and the clarified lysates were generated as described inEXAMPLE 2. HTP screening reactions were carried out as described inEXAMPLE 14, except that the lysate was diluted 16-fold before adding tothe reaction plate. Activity relative to SEQ ID NO: 1140 was calculatedas described in EXAMPLE 14.

TABLE 19.1 Relative Activities of PAL Variants on Production of Compound4 from Compound 3 (Relative to SEQ ID NO: 1140) SEQ Amino AcidDifferences FIOP (activity) ID NO: (Relative to SEQ (Relative to SEQ(nt/aa) ID NO: 1140) ID NO: 1140)¹ 1197/1198 N36Q/P47R/A424L/E517D/R554V+++ 1199/1200 K410T/E517D/R554V +++ 1201/1202 P47E/A5241 ++ 1203/1204K410Y ++ 1205/1206 A424L/E517D/R554V ++ 1207/1208 P47T/R554L ++1209/1210 P47E/L214Q/K413A/A524I/L563V + 1211/1212 E517D/A5241/R554L +1213/1214 R554L + 1215/1216 L214Q + 1217/1218 K410L/R554V + 1219/1220P47T/K410Y/A5241 + 1221/1222 L214Q/A424L + ¹Levels of increased activitywere determined relative to the reference polypeptide of SEQ ID NO:1140, and defined as follows: “+” = 1.40 to 1.60 (first 50%); “++” >1.60(next 30%); and “+++” >1.69 (top 20%)

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

We claim:
 1. An engineered polynucleotide encoding an engineeredphenylalanine ammonia lyase comprising a polypeptide sequence having atleast 95% sequence identity to SEQ ID NO: 4, wherein said engineeredphenylalanine ammonia lyase comprises a substitution at position 104 andat least one substitution selected from positions 220, 222, and 359 insaid polypeptide sequence, and wherein the amino acid positions of saidpolypeptide sequence are numbered with reference to SEQ ID NO:
 8. 2. Anengineered polynucleotide sequence encoding at least one engineeredphenylalanine ammonia lyase, wherein said polynucleotide sequencecomprises at least 85% sequence identity to SEQ ID NO: 1, 3, 7, 105,251, 445, 481, 515, 617, 713, 829, 893, 987, and/or 1139, wherein thepolynucleotide sequence of said engineered phenylalanine ammonia lyasecomprises at least one substitution at one or more positions.
 3. Theengineered polynucleotide sequence of claim 1, wherein saidpolynucleotide sequence comprises at least 85% sequence identity to SEQID NO: 1, 3, 7, 105, 251, 445, 481, 515, 617, 713, 829, 893, 987, and/or1139.
 4. The engineered polynucleotide sequence of claim 1, wherein saidpolynucleotide sequence comprises SEQ ID NO: 1, 3, 7, 105, 251, 445,481, 515, 617, 713, 829, 893, 987, and/or
 1139. 5. The engineeredpolynucleotide sequence of claim 1, wherein said polynucleotide sequencecomprises a sequence set forth in the odd-numbered sequences set forthin SEQ ID NOS: 3-1221.
 6. The engineered polynucleotide sequence ofclaim 1, wherein said polynucleotide sequence is operably linked to acontrol sequence.
 7. The engineered polynucleotide sequence of claim 1,wherein said engineered polynucleotide sequence is codon optimized. 8.An expression vector comprising at least one polynucleotide sequence ofclaim
 1. 9. A host cell comprising at least one expression vector ofclaim
 8. 10. A host cell comprising at least one polynucleotide sequenceof claim
 1. 11. The host cell of claim 10, wherein said host cell is aeukaryotic host cell.
 12. The host cell of claim 11, wherein said hostcell is Escherichia coli.
 13. A method of producing an engineeredphenylalanine ammonia lyase in a host cell, comprising culturing thehost cell of claim 10, in a culture medium under suitable conditions,such that at least one engineered phenylalanine ammonia lyase isproduced.
 14. The method of claim 13, further comprising the step ofrecovering at least one engineered phenylalanine ammonia lyase from saidculture medium and/or said host cell.
 15. The method of claim 13,further comprising the step of purifying said at least one phenylalanineammonia lyase.